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PageJournal 1 of 22 of Agricultural and Food Chemistry

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Identifying Fraudulent Natural Products: A Perspective on the Application of Carbon-14 Analysis Haley Gershon, Anna Lykkeberg, Florencia Goren, Stephany Mason 4985 SW 74th Court Miami FL, 33155 Tel: (+1) 305-662-7760 Fax: 305-663-0964 [email protected]


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Abstract

2

Across food and supplement industries, there is a growing trend towards products comprised of

3

natural ingredients. As manufacturers strive to offer “natural” products to consumers,

4

adulteration of ingredients with cheaper synthetic alternatives becomes a concern. This

5

Perspective highlights the application of carbon-14 analysis to screen for potential adulteration of

6

natural ingredients such as garlic oil. Carbon-14 testing determines if a product is comprised of

7

solely plant or animal-based ingredients by measuring the percentage of biomass versus

8

petrochemical-derived sources. Through comparison of other analytical techniques used for

9

quality control, carbon-14 testing stands out in being able to detect petrochemical-derived

10

nature-identical compounds.

11 12 13 14 15 16 17 18 19 20 21 22 23


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Introduction

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It is relatively common to encounter food products with label claims of “all-natural” or “100%

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natural” ingredients; nonetheless, it is possible that ingredients have been adulterated and are not

27

wholly derived from plant or animal sources. Often, foods are formulated to taste like a specific

28

flavor by using either natural extracts or artificial ingredients. Artificial flavors are typically

29

sourced from abundant and cheap petrochemical sources while natural flavors are usually less

30

readily available and more expensive.1 The United States Food & Drug Administration defines

31

the term natural flavor to include essential oils, essences or distillates, which contain the

32

flavoring constituents derived from, for example, a spice, fruit, vegetable, or plant material,

33

whose main function in food is for flavor rather than nutrition.2 While natural flavors are popular

34

amongst consumers, there is the potential for ingredient fraud and false label claims as natural

35

flavors can be adulterated with less expensive material.1 This is mainly due to economically

36

motivated adulteration: intentional fraudulent addition or substitution of an ingredient in a

37

product in order to increase the apparent value or decrease the cost of production, overall for

38

economic gain.3 As a result, it is vital for products to undergo quality control and quality

39

assurance steps. This perspective will focus on the application of carbon-14 testing as a tool to

40

screen “natural” products for petrochemical-derived synthetic ingredients including a case study

41

on garlic oil.

42 43

Carbon-14 Testing to Detect Adulteration

44

Natural product testing through carbon-14 analysis differentiates animal or plant-derived

45

constituents from petrochemical sources. Accelerator mass spectrometer (AMS) instrumentation

46

is able to detect very low concentrations of atoms of specific elements according to their atomic


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weight. Nowadays, AMS is the most common method used for carbon-14 measurement.4 All

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living material contains a known level of the weakly radioactive carbon isotope, carbon-14,

49

however, once a plant or organism dies, the level of carbon-14 decreases at a known rate in

50

accordance with its half-life of 5730 years. The amount of carbon-14 that is still present after

51

death is related to the amount of time since the death of the organism occurred.5 Therefore, by

52

measuring the amount of carbon-14 in a given sample, the analysis can be used not only to date

53

an archaeological or geological sample, but also to distinguish between natural biological

54

sources and fossil fuel sources, since products sourced from plants or animals have a known

55

level of carbon-14 whereas the latter is sufficiently old to be devoid of the radioactive isotope.6

56

Carbon-14 results are reported according to international standards such as American

57

Society for Testing and Materials (ASTM) D6866. ASTM D6866 is a standard test method used

58

for determining the biobased content of liquid, gaseous and solid samples by distinguishing

59

biomass-based components from fossil fuel-derived sources through the use of radiocarbon

60

analysis.7 Carbon-14 results are reported as % biobased, ranging from 0% to 100%. Per the

61

ASTM D6866 standard, all results are reported with an absolute error of +/- 3%.7 Other

62

standards, such as International Organization for Standardization (ISO) 16620-2:2015, are also

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based on carbon-14 content measurement. ISO 16620-2:2015 is an international standard used to

64

determine biobased carbon content of solid, liquid or gaseous samples that can be applied to

65

flavors, fragrances, food, beverages and supplements. ISO 16620-2 reports show the percentage

66

of a product from biobased sources versus fossil-derived constituents.8

67

Carbon-14 testing has been used in several instances to detect food adulteration where the

68

unambiguous determination of the source of an ingredient was through the measurement of a

69

product’s carbon-14 content. Calibrations of radiocarbon age determinations of natural material


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are applied to convert the conventional carbon-14 age to calendar years.9 Martin and Thibault

71

(1995) used carbon-13 and carbon-14 analyses to determine regional origin and harvest year of

72

ethanol in wines and spirits. The carbon-14 content was compared to a calibration curve to

73

indicate the year the plants were harvested.10 Starting in the early 1950s, carbon-14 analysis was

74

used for determining sources of ethanol within food products.

75

Orange juice is one of many food and beverage items that contain ethyl butyrate, a

76

popular flavor that derives from ethanol. Although ethyl butyrate can originate from natural

77

sources, the flavor can also be synthesized from petrochemical sources.1 Testing has been used to

78

authenticate natural ethyl butyrate by measuring the carbon-14 content. Byrne, Wengenroth and

79

Kruger (1986) undertook carbon-14 analysis of products containing ethyl butyrate with results

80

able to differentiate between natural and synthetic sources. Stable carbon isotope analysis was

81

applied to distinguish between different natural sources.1

82

Although the application of carbon-14 testing is consistently able to detect

83

petrochemical-derived sources, there are some limitations when using the analysis for quality

84

control purposes. Carbon-14 testing to provide balance for example for artificial vanillin is

85

limited. If synthesized from guaiacol, a petrochemical, carbon-14 will detect the adulterants.

86

However, if artificial vanillin is synthesized from natural sources like lignin, carbon-14 testing

87

will not detect the synthetic vanillin.11

88 89

Comparison of Carbon-14 and Other Methods

90

A wide range of analytical methods have been used to authenticate the natural origin of flavors

91

and other ingredients. Carbon-14 testing is often paired with other techniques to both identify a

92

variety of possible adulterants and to characterize natural or botanical-derived ingredients. Some


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93

examples include gas chromatography-mass spectrometry (GC-MS), isotope ratio mass

94

spectrometry (IRMS) and chirality-based analyses which are used on their own and in

95

combination with other analyses for quality control purposes.12 While there are a multitude of

96

analytical techniques available, this perspective is examining a few illustrative examples for

97

context but is not a comprehensive review.

98

GC-MS is an analytical technique used to determine natural constituents and is often used

99

for quality control purposes by characterizing essential oils. The gas chromatograph (GC) is used

100

to volatilize and separate the compounds of a complex mixture. The volatilized sample is carried

101

by a mobile phase (inert gas) through a stationary phase (column). Individual components of the

102

mixture interact with the chemicals in the column at different rates. This separates the

103

compounds in the mixture, resulting in a range of elution times. As the separated compounds

104

leave the GC, they enter a mass spectrometer (MS) and are broken into ionized fragments. The

105

mass to charge ratio (m/z) and relative abundance of the fragments are unique for each chemical

106

compound, providing a “fingerprint.” Since essential oils are complex matrices made up of many

107

different volatile compounds, this method is able to provide a fingerprint of the composition of

108

essential oil samples.13 GC-MS is often complemented with additional analytical techniques

109

which specifically identify the source of a material.

110

Likewise, chiral testing can also be carried out for authenticity control. When analyzing

111

essential oils, chiral testing can provide key insights regarding the purity of an essential oil. The

112

analysis is able to detect adulteration of natural products by determining whether or not synthetic

113

constituents are present. Chiral testing can take different forms, but in its essence it looks at

114

ratios of optical isomers, enantiomer ratios, specific to an essential oil. The enantiomer ratios are

115

used as markers of the natural origin of the oil, or in contrast, are used to identify adulteration as


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116

a result of mixing of material from different sources.14 When used with carbon-14 analysis, chiral

117

testing is able to detect naturally-derived material.12 Chiral testing, however, has several

118

limitations, and as a result it is not always applicable in the quality control of food ingredients

119

and flavor substances. For example, it is possible for chiral compounds to occur naturally at

120

varying enantiomer ratios. In addition, chiral odorants may be mixed in the production process of

121

natural and synthetic flavor substances.14

122

In cases where chiral analysis turns out to be insufficient, IRMS is frequently used to test

123

the source of flavors and fragrances by determining stable isotope ratios.14 IRMS measures

124

isotopic variations in natural compounds, however, if ratios are skewed, the presence of a

125

synthetic ingredient or foreign material is indicated.12 Furthermore, the combination of IRMS

126

and chiral testing is often used in authenticating citrus oils on the basis of measurements of the

127

carbon-13 to carbon-12 ratio.14

128

While GC-MS, IRMS and chiral testing are viable analyses to detect and characterize

129

natural components within a product, carbon-14 testing has the ability to detect petrochemical-

130

derived nature-identicals, synthetic compounds with the same chemical composition as the

131

natural compound found in nature.15 These differences are summarized in Table 1. Because of

132

limitations of testing methods as discussed, analytical techniques are often paired with additional

133

approaches designed to detect a variety of potential adulterants and maintain robust quality

134

control.

135 136

Adulteration of Garlic Oil

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Allium sativum, more commonly known as garlic, is widely used throughout the global arena.

138

Garlic essential oil is rich in allyl polysulfides, which make up the oil’s chemical composition.16


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With numerous applications of garlic oil segmented within the household, nutraceutical and food

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and flavors industries, the essential oil maintains a positive reputation. Garlic extracts are

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frequently used not only as a flavor during food preparation, but also as an ingredient in food

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items for digestive purposes.16 Natural garlic oil is typically prepared through steam

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distillation.17 During this process, steam is produced in a boiler and then passes through the plant

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material. Through the combination of high pressure and high temperature, the process is used on

145

a large scale for the production of essential oil. The condensed distillate consists of a mixture of

146

water and oil, in which the oil and water separate into two different layers, allowing for

147

extraction of the oil.17

148

Demand for garlic oil creates challenges for manufacturers as large quantities of raw

149

material are required for production. In order to yield a single ton of garlic oil through steam

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distillation, 500 tons of raw garlic is needed, elevating the cost of the commodity.18 Substantial

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amounts of raw material coupled with expensive garlic oil prices trigger economically motivated

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adulteration.3 Garlic oil is susceptible to adulteration and in several cases, it has been tainted

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with artificial components.19 As a result, garlic oil is vulnerable to mislabeling and fraudulent

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naturally-sourced ingredient claims as adulterated oil is marketed as “100% pure” or “all-

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natural.” In response to adulteration concerns within the garlic oil supply chain, analytical tests

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serve as a verification to ensure the authenticity of ingredients.

157 158

Garlic Oil Case Study

159

Five garlic oil samples were analyzed in a case study performed by Beta Analytic, a natural

160

product testing laboratory, in June of 2017. The purpose of the study was to screen for potential

161

petrochemical-derived adulterants in order to indicate whether sample ingredients were


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consistent with label claims. Four out of the five garlic oil samples tested included labels stating

163

“100% pure undiluted and therapeutic grade garlic oil.” The bulbs of the four Allium sativum

164

samples originated from either China or Mexico, two major growing areas of garlic. These retail

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samples were purchased from well-known online platforms in 10-milliliter bottles. The cost of

166

the garlic oils ranged, with prices between USD $3 and USD $14. The fifth sample, purchased

167

from a large chemical company, was an artificial garlic oil blend, which was included in the case

168

study for comparison purposes.20 In order to support or disprove the label claims, GC-MS, IRMS

169

and carbon-14 analyses were used with the results summarized in Table 2.

170 171

Results and Discussion

172

Through analysis performed by Aromatic Research Center, a GC-MS specialized laboratory,

173

results confirmed the chemical profile of all five garlic oil samples were consistent with the

174

chemical profile of natural garlic oil (Figure 1). According to ISO 16620-2:2015 standards, Beta

175

Analytic’s test results reported the % biobased carbon content as a fraction of total carbon

176

determined by carbon-14 content.8 Results demonstrated that only Garlic Oil #3 supported its

177

“100% pure” garlic oil label claim. The analysis was able to detect petrochemical-derived

178

synthetic adulterants present in the other three “100% pure” labeled samples, Garlic Oil #1,

179

Garlic Oil #2 and Garlic Oil #4.20 Two of the adulterated samples, Garlic Oil #1 and Garlic Oil

180

#4, had results of 0% biobased content. This indicates that the samples were derived from only

181

synthetic sources and did not contain any material sourced from plants or animals. Garlic Oil #2

182

was reported as 67% biobased, and therefore, was not purely derived from garlic. Instead, Garlic

183

Oil #2 was a combination of biomass and petrochemical sources, highlighting the reality of


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184

adulteration. Consistent with its artificial label claim, Garlic Oil #5 resulted in 0% biobased

185

carbon, in which it was entirely composed of fossil-derived inputs.

186

In conclusion, as the availability of natural-sourced products flourishes, it is increasingly

187

common to find product label claims of “100% pure” or “all-natural” ingredients. Despite the

188

claims, ingredients of natural-labeled products are vulnerable to adulteration. Because of this, it

189

is crucial for products to undergo verification for authentication purposes. The garlic oil case

190

study displays detection of fraudulent natural garlic oils available in the market. Although GC-

191

MS indicated the chemical composition of the samples matched that of natural garlic oil, the

192

carbon-14 testing identified synthetic imitations of garlic oil, underlining the importance of

193

analytical techniques to support label claims.

194 195

Future Perspectives

196

As new foods and ingredients come on the market and different market trends emerge, so do the

197

techniques and methods used for adulteration. Detecting adulteration is a complex task and there

198

is no single catch-all analytical technique that will guarantee authenticity. The future outlook for

199

the industry is therefore one of continued development, and as more sophisticated analytical

200

techniques are developed, the better the industry will be at adulteration detection. The focus of

201

this perspective, carbon-14 testing, has a specific application for natural products that are

202

vulnerable to adulteration with petrochemical-derived synthetic materials. It is especially useful

203

when the synthetic component in question is nature-identical and may not be flagged by more

204

traditional techniques like GC-MS as was shown in the garlic oil case study.


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References 1 Byrne B.; Wengenroth K. J.; Krugerand D. A. Determination of adulterated natural ethyl

butyrate by carbon isotopes. J. Agric. Food Chem. 1986, Vol. 34, No. 4, pp.736-738. 2 U.S. Food and Drug Administration; U.S. Department of Health & Human Services. CFR Code of Federal Regulations Title 21: Chapter I--Food and Drug Administration, Subchapter B-Food for Human Consumption. 2018. (https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=101.22). (18 Dec 2018). 3 U.S. Food and Drug Administration. Text Version of Randall Lutter, Ph.D. Presentation: Addressing Challenges of Economically-Motivated Adulteration. (https://www.fda.gov/NewsEvents/MeetingsConferencesWorkshops/ucm163656.htm) (3 Dec 2018). 4 Hellborg R.; Skog G. Accelerator mass spectrometry. Mass Spectrom. 2008, Rev., 27: 398427. 5 Culp R. Conformation of Botanical or Bio-Based Materials by Radiocarbon and Stable Isotope Ratio Analysis. Botanicals. Reynertson K.; Mahmood K.; CRC Press Taylor & Francis Group: Boca Raton, FL. 2015, pp. 126. 6 Buchholz B. A.; Sarachine M. J.; Zermeno P. Establishing Natural Product Content with the Natural Radiocarbon Signature. ACS Symposium Series Progress in Authentication of Food and Wine. Lawrence Livermore National Security, LLC: Livermore, CA. 7 May 2010, pp. 1. 7 ASTM International. ASTM D6866 - 18, Standard Test Methods for Determining the Biobased Content of Solid, Liquid, and Gaseous Samples Using Radiocarbon Analysis. West Conshohocken, PA, 2018. (https://www.astm.org/Standards/D6866.htm) (3 Dec 2018). 8 International Organization for Standardization. ISO 16620-2:2015, Plastics -- Biobased content -- Part 2: Determination of biobased carbon content. Geneva, Switzerland, 2015. (https://www.iso.org/standard/63767.html) (17 Dec 2018). 9 Taylor R.E.; Stuiver M.; Reimer P.J. Development and extension of the calibration of the radiocarbon time scale: Archaeological applications. Quaternary Science Reviews. 1996, Vol. 15, No. 7, pp. 655-668


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10 Martin G. J.; Thibault J. Spatial and Temporal Dependence of the 13C and 14C Isotopes of Wine Ethanols. Radiocarbon. 1995, Vol. 37, No. 3, pp. 943-954. 11 Tenailleau E. J.; Lancelin P.; Robins R. J.; Akoka S.Authentication of the Origin of Vanillin Using Quantitative Natural Abundance 13C NMR. J. Agric. Food Chem. 2004, Vol. 52, No. 26, pp. 7782-7787. 12 Tiên Do T. K.; Hadji-Minaglou F.; Antoniotti S.; Fernandez X. Authenticity of essential oils. TrAC Trends in Analytical Chemistry. 2015, Vol. 66, pp. 146-151. 13 Cordella C.; Moussa I.; Martel A.; Sbirrazzuoli N.; Lizzani-Cuvelier L. Recent Developments in Food Characterization and Adulteration Detection: Technique-Oriented Perspectives. J. Agric. Food Chem. 2002, Vol. 50, No. 7, pp. 1751–1764. 14 Zawirska-Wojtasiak R. Chirality and the Nature of Food Authenticity of Aroma. Acta Sci. Pol., Technol. Aliment. 2006, Vol. 5, No.1, pp. 30. 15 Oxford Dictionaries. Definition of nature-identical in English: nature-identical. (https://en.oxforddictionaries.com/definition/nature-identical) (3 Dec 2018). 16 Satyal P.; Craft J. D.; Dosoky N. S.; Setzer W. N. The Chemical Compositions of the Volatile Oils of Garlic (Allium sativum) and Wild Garlic (Allium vineale). Foods. 2017, Vol. 6, No. 8, pp. 1. 17 Kubeczka K. History and Sources of Essential Oil Research. Handbook of Essential Oils Science, Technology and Applications, Edition 1; Baser K. H. C.; Buchbauer G. CRC Press Taylor & Francis Group: Boca Raton, FL, 2010; pp. 5. 18 Transparency Market Research. Garlic Oil Market - Global Industry Analysis, Size, Share, Growth, Trends, and Forecast 2017 - 2025. URL (www.transparencymarketresearch.com/garlicoil-market.html) (3 Dec 2018). 19 Garside J. A Closer Look at Garlic Oil: Verifying Naturality. (https://www.perfumerflavorist.com/flavor/research/A-Closer-Look--436985963.html) (18 Dec 2018). 20 Garside J.; Goren F.; Lykkeberg A. Quality Assurance Testing: Is your Garlic Oil Natural? (https://www.nutraceuticalsworld.com/blog/blogs-and-guest-articles/2017-11-08/qualityassurance-testing-is-your-garlic-oil-natural/52030) (18 Dec 2018).


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Tables Table 1 Common analytical methods for the authenticity of natural products and the type of adulterants the analyses can detect. Table 2 Garlic Oil Case Study Results Figures Figure 1 Chromatograms and compositional analysis of the five tested garlic oils analyzed by GC-MS


12

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Table 1. Common analytical methods for the authenticity of natural products and the type of adulterants the analyses can detect. Detectable Adulterants

Analysis

Petrochemical-derived Nature-identical

Natural Ingredients

Gas Chromatography-Mass Spectrometry

✖

✔

Chiral Testing

✖

✔

Isotope Ratio Mass Spectrometry

✖

✔

Carbon-14 Testing

✔

✖


13


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Table 2. Garlic Oil Case Study Results Sample Code

Botanical Name

Label Claim

GC-MS

Biobased %

Percent modern carbon (pMC) Value

Disintegrations per minute per gram (dpm/g)

δ13C IRMS Values

Garlic Oil #1

Allium sativum

Pure Garlic Essential Oil 100% Pure Therapeutic Grade Steam Distilled Mexico

Garlic Oil PASS

0%

Identifying Fraudulent Natural Products: A Perspective on the

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