Addressing adulteration with stable isotopes.
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1 Addressing adulteration with stable isotopes. March 2008
2 Key application areas of stable isotopes Food Authentication Flavours, wine, juices, honey adulteration Environmental Science Agriculture, Forestry and Ecology Plant, litter, soil, filters, respired CO 2, etc. Archaeology and Anthropology Wildlife Tracking Atmospheric Chemistry Source apportionment of greenhouse gases Earth Science Deuterium tracing, oil/oil correlation, gas sourcing, carbonates Climatic change, oceanic-atmospheric interactions Medical Research Mass balance, blood bicarbonate, breath Metabolism, doubly labelled water, total body water Gastric emptying, liver function Protein turnover Forensics Drug doping, drug sources
3 Food Adulteration: Why and How? Main type of Adulteration - The authenticity of food relates to information on name, ingredients, origin or processing. - Frauds include undeclared addition of water or other inexpensive materials (adulteration) and false statements about sources of ingredients (i.e. geographic, plant or animal origin) - Increase profit and unfair competition for honest producer. - Cheating Consumer. Isotopic Tools - Adding water can be detected by 18 O measurements - Adding sugar can be detected by 13 C, 18 O or 2 H measurements - False origin (multi element isotopic approach; C, N, H, & S) - As it is not commercially viable to circumvent isotopic detection, stable isotopes have provided a way by which these types of frauds can be detected - Isoprime IRMS - Inlets interface such as GC, EA or LC
4 Isotopic fractionation
5 Isotopic fractionation of carbon isotopes in plants Decreasing 13 C content
6 Authenticity of.. Fruit juice Vanillin
7 Authenticity of Juice The most common type of fraud is extension of the juice by adding sugars and water Water added to mask addition of concentrated juice Cane, corn or beet sugar can be use
8 Carbon Isotope Ratio Analysis of Juice Can be used to detect the addition of C4 sugars e.g. cane and corn syrups to juice Key limitation Not possible to distinguish between the sugars in apple or orange juice and those from beet sucrose
9 Stable carbon isotope ratio analysis of solid phase δ 13 C range Reference Apple Juice to Rossmann et al (1990) Orange Juice to Bricout & Koziet (1987) Corn Syrup -9.5 to Rossmann et al (1990) Cane Sugar to Bricout & Koziet (1987) Beet Med Invert Syrup to Kelly et al (1994) Beet Sugar to Kelly et al (1994)
10 Factors affecting D/H ratio Evapotranspiration from plants Differing rates of evapotranspiration between ground (beet) and aerial plants (apple trees) give rise to relatively enriched apple sugars H 2 O DHO H 2 O DHO
11 APPLE JUICE Preparation method to determine the 2 H/ 1 H ratio of the carbon bound hydrogens in fructose FILTER ION EXCHANGE HPLC separation of fructose particulates Acids and minerals SYNTHESIS OF HMT FROM FRUCTOSE Py-GC-IRMS ANALYSIS OF HMT 1 day for preparation of hexamethylene and IRMS analysis
12 Typical Ion Chromatogram of HMT and an Internal Standard 7.1e e-012 Reference H 2 peaks m/z 2 m/z 3 HMT n-hexadecane IS 4.6e e Time / seconds
13 Rapid detection of added sugar in apple juice using hexamethylenetetramine
14 Comparison of D/H and 13 C/ 12 C ratios for, Beet Sugar, Cane Sugar and Apple Juices HMTδ 13 C V-PDB Beet 2 H C < 9 Cane Apple HMT δ 2 H V-SMOW England W. Europe Germany Ukraine Australia & NZ S. Africa USA & Canada Brazil & Chile E. Europe BEET CANE
15 Effect of controlled adulteration of apple juice y = x R 2 = % BMIS
16 Multielement IRMS approach origin assignment and authenticity check of food, discrimination between convential and organic production, identification of recent and prehistoric nutrition bases, ecological, biological and environmental investigations?
17 Authenticity of. Fruit Juice Vanilin
18 Typical combustion data for Vanillin COMBUSTION SAMPLE δ13c PDB CO2 (45/44) VANILLIN (LIGNIN) VANILLIN (GUAIACOL) VANILLIN C VANILLIN (NATURAL) -20.1
19 Comparison of pyrolysis and combustion data COMBUSTION PYROLYSIS SAMPLE δ13c PDB CO2 (45/44) δ13c PDB CO (29/28) VANILLIN (LIGNIN) VANILLIN (GUAIACOL) ± ± 0.3 VANILLIN C ± 0.2 VANILLIN (NATURAL) ± 0.3
20 Vanillin substituent specific δ 13 C H δ13caverage PDB δ13cring = PDB (estimated) H C=O C O δ13ccarbonyl = PDB H δ13cmethyl = PDB O C H H
21 δ 13 C,δ 18 O and δ 2 H data for Vanillin samples COMBUSTION PYROLYSIS SAMPLE δ 13 C PDB CO 2 (45/44) δ 13 C PDB CO (29/28) δ 18 O VSMOW CO (30/28) δ 2 H VSMOW H 2 (3/2) VANILLIN (LIGNIN) VANILLIN (GUAIACOL) ± ± ± ± ± ± 3.1 VANILLIN C ± ± ± 0.3 VANILLIN (NATURAL) ± ± ± 1.1
22 PY-CF-IRMS Rapid continuous flow method Allows determination of 2 H abundance in organics δ 18 O and δ 13 C substituent specific measured simultaneously Rapid detection of isotopically manipulated vanillin
23 IRMS Specificity Product bar-coding SIR MS Identification? Quantification? Origin? GC MS LC MS Exact mass Intact product / impurity identification
24 IsoPrime-Benchtop IRMS A true benchtop stable isotope mass spectrometer An easy addition to any laboratory
25 IsoPrime-Analyser Horizontal flight tube 90 º asymetric geometry Effective magnetic deflection of 108mm Turbomolecular pump mounted under source block Analyser housing can be baked to 120 o C (bake-out heaters fitted as standard for dual inlet systems).
26 Sample line Open Split Prep. He carrier Time Time System Mass Spec Reference gas supply He carrier Time Reference line
27 CO 2 Analysis
28 Hydrogen Analysis Extra bucket (mass 2) is used with the wide (46) bucket
29 MultiFlow-Bio 2 H*, 18 O Waters, Urea, juice, wine * CF-H 2 Sample Size 200 μl Specification δ 18 O: 0.15 δ 2 H: 2.0
30 Equilibration with the MultiFlow δ 2 H: He + 10% H 2 δ 18 O : He + 5% CO 2 IRMS Pt catalyst for H 2 only 200 ml of sample 40 C 90 mins. Analysis Time = 1.5 minutes/sample
31 MultiFlow Water removal TCD 4-way valve 6-way valve Column Triple core needle M.S. Autosampler Acid pump
32 GC-Combustion Interface Specification δ 13 C: 0.2 δ 2 H: 4.0
33 GC-IRMS COMBUSTION 13 C, 15 N analyses from mixtures separated by GC and combusted to CO 2 and N 2 PYROLYSIS D/H, analyses from mixtures separated by GC and pyrolysed to H 2
34 GC-Combustion Interface Key Features Heart-split mechanism combustion of all, or sections of, the GC effluent High efficiency Combustion Furnace production of CO 2, N 2, H 2 O (cf. methane) ChromeHD TM technology unique technology for production of H 2 for GC-pyrolysis Water trapping efficiency membrane or cryogenic traps Calibration methods internal reference compound (usually deuterated) external reference gas (Reference Gas Injector) Background subtraction software
35 Elemental Analyser: vario MICRO cube Optimized for micro samples Fully digitalized Simultaneous CNS (IRMS) or CHNS (TCD) Low Blank auto sampler
36 Functional Diagram of Simultaneous CHNS Stable Isotope and Elemental Analysis
37 How to Perform Multielement IRMS? I) Conventionally: Three samples for the IRMS of four elements 13 C, 15 N: Combustion, GC separation of N 2 and CO 2 ; IRMS 2 H, ( 18 O): Pyrolysis, GC separation of H 2 and CO (in absence of N); IRMS 34 S: Combustion, GC separation of SO 2 ; IRMS II) By our system: One sample for the IRMS and EA of four elements 13 C, 15 N, 2 H, 34 S: Combustion, adsorptive separation of all gases; reduction of H 2 O; IRMS
38 Ongoing - GC-IRMS Detection of Fruit Juice Adulteration EA- Method GC-Method - Juice sample Centrifugation (2000*g) + Filtration Juice Sample Solids removed Centrifugation (2000*g) and Filtration Cation and Anion Exchange Solids removed Rotary evaporation Lyophilization of aqueous fraction Semi- preparative LC 1 mg sample + methylboronic acid in pyridine Collection of individual sugars + BSTFA (after 30 mins ) Lyophilisation of sugars Determine δ13 C of individual sugars by GC-IRMS EA IRMS δ 13 C of individual sugars NEW LC Interface
39 Liquid Chromatography/IRMS LiquiFace Dionex ICS 3000 Sugars Amino acids Organic acids
40 Schematic of the LC Interface Dissolved material converted to gaseous form by reaction with suitable oxidizing agents Then passed through heater at 98 ºC to speed up conversion to CO 2 sparge gas chemicals The cooler returns the solution to room temperature to reduce vapor pressure of water Cooling the solution minimizes the passage of water vapor through membrane The He/CO 2 then passes through a Nafion drier and finally into MS HPLC to Isoprime pump manual mixing injection tee valve pump nafion heater cooler gas exchanger waste He in
41 IonVantage software IsoPrime: The True XP IRMS Sample list IsoPrime Instrument control Dual Inlet control Advanced settings
42
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