WP4: DETECTION OF WATER ADDITION IN NFC JUICES · Orange juice -single strength Spain 14 2003/2004 Orange juice -single strength Morocco 2 2003 ... Glucose + fructose. 25 27 29 31

PROJECT: GRD1-2001-41818 “PURE JUICE”

“DETECTION AND PREVENTION OF ADULTERATION ON THE EU FRUIT JUICE MARKET BY INVESTIGATION OF THE ISOTOPIC AND COMPOSITIONAL PROFILE OF MINOR COMPONENTS”

WP4: DETECTION OF WATER ADDITION IN NFC JUICES

Darmstadt, 9th March 2006

Ministerio de Agricultura, Pesca y Alimentación. Madrid (Spain)

OBJECTIVES

To establish an oxygen isotopic data base for authentic fruit juice

To improve the detection of water addition in single-strength juices:

•Optimization isotopic methods and extraction procedures(organic compounds of juices)

•Investigation correlations between 18O values of water and other components of the juices

20041SpainLemon juice, single strength

20031BelizeWhite Grapefruit juice, single strength

20031USARed Grapefruit juice, single strength

20041IsraelRed Grapefruit juice, single strength

20032SpainMandarin juice, single strength

2003/200414SpainOrange juice - single strength

20032MoroccoOrange juice - single strength

2002/20049MexicoOrange Juice - single strength

2003/20045ItalyOrange juice, single strength

20034GreeceOrange juice - single strength

20034USAOrange juice - single strength

2002/20033CubaOrange Juice - single strength

2002/200414BrazilOrange Juice - single strength

20021BelizeOrange Juice - single strength

20031ArgentinaOrange juice - single strength

Year of harvestnCountryProduct Description

SAMPLES

2%

3%

22%5%

8%

6%

8%

14%

3%27%

2%

A rgentina

Belize

Brazil

Cuba

USA

Greece

Italy

Mexico

Morocco

Spain

Israel

63 authentic juice samples

Isotopic analysis

ISOLATION OF SUGARS

Ø CEN : ENV 12140 method (precipitation and acidification)

Ø Anion exchange resin column

Ø Semipreparative HPLC system

0.5SD

30.6Mean δ18O pulp (‰)

30.331.430.330.930.3Mean

30.231.030.630.331.3Run 4

30.831.130.030.931.0Run 3

30.331.830.730.929.5Run 2

29.731.829.931.329.2Run 1

δ18O pulpR033091

(5)(‰)

δ18O pulpR033091

(4)(‰)

δ18O pulpR033091

(3)(‰)

δ18O pulpR033091

(2)(‰)

δ18O pulpR033091

(1)(‰)

0.30.30.3SD

31.031.130.7Mean

31.130.830.3

31.431.230.6

30.630.630.4

30.731.030.4

31.131.231.0

31.431.731.0

31.030.931.3

30.431.431.0

31.031.530.5

δ18O cellulose(‰)

Day 3


Day 2


Day 1

18O/16O ANALYSIS OF ORGANIC COMPOUNDS TC/EA furnace coupled on line with a Finnigan DELTA plus Spectrometer

Oven temperature 1425 ºC and tin capsules

EXTRACTION AND PURIFICATION OF THE PULP

CEN : ENV 13070 method

18O/16O ANALYSIS OF WATER OF AUTHENTIC SAMPLES

Finnigan Delta S SpectrometerCO2 after equilibration with the juice water according to the CEN:ENV 13141:1997 method

Repeatability and reproducibility

ISOLATION OF SUGARS

Centrifugation (5.500 rpm, 10 min)

Pass through C18 Sep-pak cartridge

Filter through a 0.45 μm filter

HPLC (Waters Spherisorb S5 NH2)Anion-exchange resin (3 ml of AG1-X8, 100-200 mesh)Mixed resin (3 ml of AG501-X8, 20-30 mesh)

- glucose + fructose- sucrose

LiophylisationLiophylisation

ANION EXCHANGE RESIN SEMIPREPARATIVE HPLC

δ 18O wate r (‰ )

-4

-2

0

2

4

6

8

Argent

ina

Belize

Brazil

Cuba

USA

Gre

ece Italy

Mexico

Moro

cco

Spain

Country

δ 18O wate r (‰ )

-4

-2

0

2

4

6

8

Argent

ina

Belize

Brazil

Cuba

USA

Gre

ece Italy

Mexico

Moro

cco

Spain

Country

δ18O ISOTOPE VALUES OF WATER FROM SINGLE STRENGTH ORANGE JUICE SAMPLESOF DIFFERENT ORIGINS

18O/16O ANALYSIS OF WATER

1.14.51.12.814Spain

-0.47.31.82.953Total

3.46.72.35.12Morocco

1.14.71.12.69Mexico

-0.31.60.90.95Italy

-3.9-0.91.3-2.04Greece

2.13.20.52.74USA

3.65.00.74.23Cuba

0.36.52.33.814Brazil

------3.21Belize

-------0.41Argentina

Minimumδ18O water

(‰)Maximum δ18O

water (‰)SDMean δ18O water (‰) nCountry

MEAN

0

10

20

30

40

Percentage (%)

[0/1] [1/2] [2/3] [3/4] [4/5]

18O Wate r

Spain

0

20

40

60

Perce ntage (%)

[-1/0] [0/1] [1/2]

18O Water

Italy

WATER OXYGEN DISTRIBUTION

0

5

10

15

20

25

Percentage (%)

[-1/0] [0 /1] [ 1/2 ] [ 2 /3 ] [ 3 /4] [4 /5] [ 5/6 ] [ 6 /7] [ 7/8]

18O Water

0

10

20

30

Percentage (%)

[0/1] [2/3] [4/5] [6/7]

18O Water

Brazil

0

10

20

30

40

Percentage (%)

[1/2] [2/3] [3/4] [4/5]

18O Water

Mexico

98%> 0

87% > 2

Included withinthe AIJN values

86% > 2

78% > 2

Not included withinthe AIJN values

26.831.01.528.914Spain

23.231.02.126.753Total

30.130.10.130.12Morocco

23.226.21.025.19Mexico

26.228.81.127.15Italy

27.430.21.328.34Greece

23.926.91.325.84USA

24.726.10.825.23Cuba

23.228.41.425.714Brazil

------23.51Belize

------25.21Argentina

Minimumδ18O pulp(‰)

Maximumδ18O pulp(‰)

SDMean

δ18O pulp (‰)nCountry

δ18O ISOTOPE VALUES OF PULP FROM SINGLE STRENGTH ORANGE JUICE SAMPLESOF DIFFERENT ORIGINS

δ 18O pulp (‰ )

0

5

10

15

20

25

30

35

Argent

ina

Belize

Brazil

CubaUSA

Gre

eceIta

ly

Mex

ico

Mor

occo

Spain

Country

δ 18O pulp (‰ )

0

5

10

15

20

25

30

35

Argent

ina

Belize

Brazil

CubaUSA

Gre

eceIta

ly

Mex

ico

Mor

occo

Spain

Country

18O/16O ANALYSIS OF PULP

MEAN

δ 18O sugar (‰ )

0

5

10

15

20

25

30

35

40

Argentin

a

Belize

Brazi

l

CubaUSA

Gre

eceIta

ly

Mex

ico

Mor

occo

Spain

Country

δ 18O sugar (‰ )

0

5

10

15

20

25

30

35

40

Argentin

a

Belize

Brazi

l

CubaUSA

Gre

eceIta

ly

Mex

ico

Mor

occo

Spain

Country

δ18O ISOTOPE VALUES OF SUGAR FROM SINGLE STRENGTH ORANGE JUICE SAMPLESOF DIFFERENT ORIGINS

33.036.11.334.57Spain

30.437.51.833.346Total

36.737.50.537.12Morocco

30.634.81.332.08Mexico

31.033.11.032.75Italy

31.734.01.033.14Greece

------32.61USA

33.534.40.433.93Cuba

31.535.51.333.614Brazil

------31.01Belize

------30.41Argentina

Minimumδ18O sugar (‰)

Maximumδ18O sugar (‰)

SDMean

δ18O sugar (‰)nCountry

18O/16O ANALYSIS OF SUGARS

MEAN

ISOLATION OF SUGARS

35.6

32.0

32.6

32.0

35.5

35.3

34.6

33.4

32.5

30.4

32.2

31.7

32.5

37.5

36.7

33.5

30.6

34.0

32.9

31.7

33.6

31.5

35.9

34.9

δ18O sugar (‰)

33.8

32.1

32.3

32.1

34.1

34.1

35.1

34.1

32.2

31.6

30.6

34.5

32.0

36.2

36.4

n.m

29.3

34.4

31.8

32.9

33.0

29.8

35.5

36.0

δ18O sucrose

(‰)

30.8SSOS 11119-04

28.3SSOS 14050

26.1SSOS 15547

28.3SSOS 14050

31.0SSOS 13589

30.7SSOS 13574

33.8SSOS 13562

31.4SSOS 13113

31.5SSOS 13097

29.7SSOS 12373

30.1SSOS 11394

33.6SSOS 11201

29.3SSOS 11153

30.6SSOS 11060

34.3SSOS 11050

29.7SSOS 11029

27.8SSOS 10959

31.2SSOS 10582

34.3SSOS 10578

34.2SSOS 10568

33.6SSOS 10552

27.7SSOS 10141

30.9SSOS 10357

30.3SSOS 10485

δ18O (glucose + fructose)

(‰)

Sample

y = 0.8192x + 5.6518

R2 = 0.7827

20

25

30

35

40

30 32 34 36 38

18O Sucrose

18O

To

tal

Su

gar

SEMIPREPARATIVE HPLC

MV

0.00

100.00

200.00

300.00

400.00

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Sucrose

Glucose + fructose

25

27

29

31

33

35

37

39

-1 1 3 5 7 9

O water

O s

ug

ar

Argentina

Belize

Brazil

Cuba

USA

Italy

Mexico

Morocco

Spain

y=0.6008x + 31.419

R= 0.64

CORRELATION OF δ18O-VALUES OF SUGAR AND WATER IN ORANGE JUICES

20

22

24

26

28

30

32

-1 1 3 5 7 9

O w ater

O p

ulp

Argentina

Belize

Brazil

Cuba

USA

Italy

Mexico

Morocco

Spain

y=0.0743x +26.452

R= 0.006

CORRELATION OF δ18O-VALUES OF PULP AND WATER IN ORANGE JUICES

32.125.8-0.114050-03 (20% water)

34.628.2-0.910107-03 (20% water)

34.628.40.810107-03

32.326.2n.m.14050-03 (20% water + concentrate juice)

32.325.71.814050-03

34.126.12.313719 (20% water + concentrate juice)

33.225.32.213719 (20% water)

32.725.84.713719

37.832.22.216078-03 (20% water)

37.732.04.716078-03

δ18O sugar(‰ )

δ18O pulp(‰ )

δ18O water(‰ )

Sample

WATER ADDITION

NOT MODIFIED

Sample OS 12

-24,0-24,633,927,22,3SpainOS 16

-23,9-24,733,127,30,8SpainOS 15

-24,1-25,734,327,02,2MexicoOS 14

-23,4-24,433,927,30,8SpainOS 13

-24,1-25,932,225,72,3FloridaOS 12

δ13C sugar(‰ )

δ13C pulp(‰ )

δ18O sugar(‰ )

δ18O pulp(‰ )

δ18O water (‰ )

OriginSample Number

EVALUATION OF FIVE ORANGE JUICES

Øδ18O (‰ ) values of water, pulp and sugar are included within the range (Floridian Pure Juice Project values) Øδ13C (‰ ) values of pulp and sugar are within the AIJN parameters

13C pulp-13C sugar (-1.8) is not included in the -1/+0.5 accepted intervalδ13C values of pulp are not included within the range (Floridian Pure Juice Project values)

The sample can not be considered adulterated by water addition. Nevertheless, a manipulation could be considered

Øδ18O (‰ ) values of pulp and sugar are included within the range (Spanish Pure Juice Project values)the δ18O value of water is not included within the range

Considering the values of the 14 Spanish samples from the Pure Juice project (years 2003 and 2004):% Water= 2.8-0.8 x 100=19%

2.8+7.5

Øδ13C (‰ ) value of sugar (-23.9) is not included in the AIJN accepted interval (-24/-27)

ØThe sample shows a 19% water addition (considering Pure Juice project data) and a sugar addition can be detected

Sample OS 13

-24,0-24,733,927,22,3SpainOS 16

-23,9-24,733,027,30,8SpainOS 15

-24,1-25,734,327,02,2MexicoOS 14

-23,4-24,433,927,30,8SpainOS 13

-24,1-25,932,225,72,3FloridaOS 12

δ13C sugar(‰ )

δ13C pulp(‰ )

δ18O sugar(‰ )

δ18O pulp(‰ )

δ18O water (‰ )

OriginSample Number


Sample OS 14


Ø18O (‰ ) values of water and sugar are included within the range (Mexican Pure Juice Project values)Øδ18O values of pulp are not included within the range (26.6-23.2)Øδ13C (‰ ) values of pulp and sugar are within the AIJN parameters

13C pulp-13Csugar (-1.7) is not included in the -1/+0.5 accepted intervalδ13C values of pulp are not included within the range (Mexican Pure Juice Project values)

The sample can not be considered adulterated by water addition. Nevertheless, a manipulation could be considered

-24,0-24,733,927,22,3SpainOS 16

-23,9-24,733,027,30,8SpainOS 15

-24,1-25,734,327,02,2MexicoOS 14

-23,4-24,433,927,30,8SpainOS 13

-24,1-25,932,225,72,3FloridaOS 12

δ13C sugar(‰ )

δ13C pulp(‰ )

δ18O sugar(‰ )

δ18O pulp(‰ )

δ18O water (‰ )

OriginSample Number

Sample OS 15


Øδ18O (‰ ) values of pulp and sugar are included within the range (Spanish Pure Juice Project values)the δ18O value of water is not included within the range

Considering the values of the 14 Spanish samples from the Pure Juice project:% Water= 2.8-0. 8 x 100=19%

2.8+7.5

Øδ13C (‰ ) value of sugar (-23.9) is not included in the AIJN accepted interval (-24/-27)

ØThe sample shows a 19% water addition (considering Pure Juice project data) and a sugar addition can be detected

-24,0-24,733,927,22,3SpainOS 16

-23,9-24,733,027,30,8SpainOS 15

-24,1-25,734,327,02,2MexicoOS 14

-23,4-24,433,927,30,8SpainOS 13

-24,1-25,932,225,72,3FloridaOS 12

δ13C sugar(‰ )

δ13C pulp(‰ )

δ18O sugar(‰ )

δ18O pulp(‰ )

δ18O water (‰ )

OriginSample Number

Sample OS 16


Øδ18O (‰ ) values of water, pulp and sugar are included within the range (Spanish Pure Juice Project values)Øδ13C (‰ ) value of sugar and pulp are included within the range and the AIJN accepted interval (-24/-27)

The sample can not be considered adulterated by water addition

-24,0-24,733,927,22,3SpainOS 16

-23,9-24,733,027,30,8SpainOS 15

-24,1-25,734,327,02,2MexicoOS 14

-23,4-24,433,927,30,8SpainOS 13

-24,1-25,932,225,72,3FloridaOS 12

δ13C sugar(‰ )

δ13C pulp(‰ )

δ18O sugar(‰ )

δ18O pulp(‰ )

δ18O water (‰ )

OriginSample Number


ØAIJN parameters were not able to detect water additionØSpanish DB let water addition detection

No addition

19% water

manipulation

19% water

manipulation

Addition detected

-24,0

-23,9

-24,1

-23,4

-24,1

δ13C sugar(‰ )

No addition-24,733,927,22,3SpainOS 16

10% water-24,733,027,30,8SpainOS 15

18% water-25,734,327,02,2MexicoOS 14

10% water-24,433,927,30,8SpainOS 13

5% water-25,932,225,72,3FloridaOS 12

Additionδ13C pulp

(‰ )δ18O sugar

(‰ )δ18O pulp

(‰ )δ18O water

(‰ ) Origin

Sample Number

SAMPLE δ13C (‰ ) (D/H)I (D/H)II

KSAS45439 -26.5 96.4 121.3

KSAS45559 -28.6 96.3 122.2

δ13C of derived CO2 of ascorbic acid degradation

(‰ )

δ13C ascorbic acid (‰ ) Ascorbic acid Ascorbic acid + Na2SO4

1st trial

2nd

trial Mean 1st trial

2nd

trial Mean 1st trial

2nd

trial Mean

CR

681/03 -16.4 -16.3 -16.4 -19.6 -21.6 -20.6 -23.2 -23.3 -23.3

SA

MP

LE

CR

856/03 -21.4 -21.6 -21.4 -27.0 -27.1 -27.0 -28.6 -29.2 -28.9

-33

-32

-31

-30

-29

-28

-27

-26

-25

160 200 240 280

Time (min)

13

C trial 1

trial 2SAMPLE δ13C total malic

(‰ )

δ13C C1 from malic

(‰ )

1 0172-0540 -26,8 -27,7

2 0172-0541 -27,6 -30,6

3 0172-0542 -26,7 -27,7

4 0172-0543 -27,6 -30,8

5 0172-0544 -25,9 -24,7

6 0172-0545 -25,8 -24,7

SAMPLE δ18O (‰ )

δ18O after

dehydratation

(‰ )

0166/3776 16.9 27.0

0166/4072 13.8 23.1

COLABORATION WITH OTHER LABORATORIES

Not detected52ASSOS-10478-03









HPLC

(DL 8mg/l)

Enzymatic

mg/l

Sample Number

1. Interlaboratory collaborative study for 13C, (D/H)I and (D/H)II measurements on alcohol

2. Pre-ring test: analysis of 13C at the C1 position of ascorbic acid

3. A peer study of a positional 13C-IRMS method for the detection of L-malic acid addition to apple juice

4. Interlaboratory collaborative study for 18O measurement on alcohol and Sorbitol determination

5. 18O water measurement on fruit juices

1

2 34

5

6

1-Inositol

2-Xilitol

3-Sorbitol

4-Manitol

5-Fructose

6-Glucose

13%

16%

8%13%4%

13%

16%17%

Brazil Costa Rica Honduras Indonesia

Kenya Philippines South A f rica Thailand

-0.12004ThailandSSAN 15891-04

1.12004ThailandSSAN 15853-04

2.62004South AfricaSSAN 15306-04


-4.32004PhilippinesSSAN 15098-04


-3.02004IndonesiaSSAN 13533-04



-3.12004Cost RicaSSAN 12352-04




2.12002IndonesiaSSAN 15972-02

1.32004HondurasSSAN 11305-04

1.22003BrazilSSAN 16695-03

0.72004KenyaSSAN 10291-04



1.02003HondurasSSAN 16257-03


6.12003BrazilSSAN 16010-03

0.82003IndonesiaSSAN 15559-03

7.22003BrazilSSAN 13128-03

δ18O water(‰ )

Year of harvest

CountrySample Number

0

5

10

15

20

25

30

35

Percentage (%)

[-7/ -6] [-6/ -5] [-5/ -4] [-4/ -3] [-3/ -2] [-2/ -1] [-1/ 0] [0/ 1] [1/ 2] [2/ 3] [3/ 4] [4/ 5] [5/ 6] [6/ 7] [7/ 8]

18O water

Pineapple

PINEAPPLE SAMPLES

Not included within the AIJN parametersDatabase is needed to evaluate properly

Brazil and Mexico “false water addition”(14% and 22% samples with δ18O <2)

CONCLUSIONS

Oxygen data base

Established for authentic single strength orange juice

70% samples from 3 countries (Brazil, Mexico and Spain)

an statistical analysis cannot be carried out

Pulp and sugar

Extraction procedures and isotope methods optimized

good reproducibility, repeatability, vc

Pulp and sugar same magnitude, but are not correlated

to 18O values of water

Further studies are required to establish an internal reference to detect

water addition at lower levels than method based on the water only

2/3 CO2 atmosphere

AIJN code Blind samples no water addition detection

Pure Juice DB detected water addition in Spanish samples

A revision of AIJN code is proposed

THANK YOU

FOR YOUR ATTENTION

Documents

WP4: DETECTION OF WATER ADDITION IN NFC JUICES · Orange juice -single strength Spain 14 2003/2004 Orange juice -single strength Morocco 2 2003 ... Glucose + fructose. 25 27 29 31