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GRD1-2001-41818 PURE JUICE

Pure Juice

WP 2 Detection of sugar addition in fruit juice

Dr. Paul Reece


FRUIT JUICE

Major Components

Minor Components

Pulp Acids Otherwater Sugars


FRUIT JUICE

Major Components

Minor Components


12C/13C

Fermentationto ethanol

12C/13C 2H/1H


FRUIT JUICE

Major Components

Minor Components


12C/13C


12C/13C 12C/13C2H/1H 2H/1H

PeriodateDegradation of

fructose to HMT


FRUIT JUICE

Major Components

Minor Components


12C/13C


Separationglu, fru,suc

12C/13C 12C/13C 12C/13C2H/1H 2H/1H

PeriodateDegradation of

fructose to HMT


WP2 - Objectives

• T2.2 Optimisation of sugar isolation by semi-prep HPLC

• T2.3 δ13C measurement on isolated sugars

• T2.4 Determination of δ2H and δ13C in HMT derived from isolated sugars

• T2.5 Investigate extending methods to sorbitol for use as internal isotopic reference

• T2.6 Validation and peer testing


T2.2 Optimisation of sugar isolation by HPLC

Crude sugar fraction obtained by either:

•pre-treatment of juice with CaOH

•using Ion exchange purification

Comparison of HPLC profiles of individual sugars from a number of juices using both methods showed loss of glucose and fructosewith CaOH extraction procedure.


0

200

400

600

800

1000

1200

0 10 20 30 40 50 60

0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 60

Calcium precipitation

0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 60

0

50

100

150

200

250

300

350

0 10 20 30 40 50 60

Ion exchange Jamin et al

Calcium precipitation

Ion exchange Jamin et al

Pineapple juice sample Orange juice sample

KSOS 10304KSAN 15973

Fructose glucose sucrose


Optimisation of sugar isolation by HPLC –column type

Polymer Labs Ca 2+ Carbohydrate Column

Time / minutes

0 5 10 15 20

De

tect

or

Ou

tpu

t / m

V

0

100

200

300

400

500

Sugar standards

Time / minutes

0 5 10 15 20

De

tect

or

Sig

na

l / m

V

0

200

400

600

800

Pineapple Juice (12 Brix)

•increase in background - possibly retained sugars•retention volumes gradually increase with no. of injections

Sucrose

glucose

fructose


•no increase in baseline •retention volumes consistent even after intensive operation

YMC Polyamine Column

Optimisation of sugar isolation by HPLC –column type (cont.)

Time / minutes

0 20 40 60

De

tect

or

Sig

na

l / m

V

-50

0

50

100

150

200

250

300

350

sugar standards

Time / minutes

0 20 40 60

De

tect

or

Sig

na

l / m

V

0

50

100

150

200

250

300

350

pineapple juice (12 Brix)

fructoseglucose

sucrose


Conclusions to task T2.2:Optimisation of sugar isolation by HPLC

• Ion exchange purification of juice followed by Polyamine HPLC separation provides most reliable separation of individual sugars


13C ratio of Orange Juice 13C ratio of Pineapple Juice(KSOS 10304) (KSAN 15973)

TOTAL SUGARS

Ca OH Ion exchange Ca OH Ion exchange

-25.48 - 24.0 -12.67 -11.46

FRUCTOSE nd - 26.9 nd -14.24

GLUCOSE nd - 24.29 nd -12.73

SUCROSE - 24.08 - 23.32 -12.62 -11.01

T 2.3 δ13C measurement on isolated sugars

-25.0-26.3-27.0 -25.3

-25.1-26.5-26.8KSAS 14280-03

-24.6-26.0-26.3

-25.4-24.1-26.4-27.1

KSAS 14520-02

-24.1-23.5-25.4-26.5

KSAS 14043-02

-25.5

-25.1

-24.5

-24.2

-23.1

δ13Cs

-25.8

-26.3-27.0KSAS 11249-04

-26.5-27.5

-26.2-26.5

-25.1-25.9-26.8

KSAS 14788-03

-25.1-25.4

δ13Ctotalδ13Cgδ13CfSample (Apple)

Ca 2+ carbohydrate column100% water mobile phase90oC,1 ml /min

Polyamine column82% acetonitrile/18% watermobile phase, 25oC10 mL / min


GC-IRMS derivatisation

Minimisation of carbon addition was accomplished by derivatisationwith methylboronic acid (MBA) and TMS thereafter.

Monosaccharides derivatized using the MBA method instead of TMS derivatives reduced the number of added carbon atoms from 2.2-2.7 to 0.3-0.8 per sugar carbon atom.

Independent measurements of δ13C of the individual sugars


δ13C Measurement of Individual sugars

- -EA- Method

Juice sample

Centrifugation (2000*g) + Filtration

Cation and Anion Exchange

Rotary evaporation

Semi-preparative LC

EA–IRMS δ13C of individual sugars

Solids removed

Collection of individual sugars

Lyophilisation of sugars

GC Method Juice Sample

Centrifugation (2000*g) and Filtration

Lyophilization of aqueous fraction

Solids removed

1 mg sample + methylboronic acid in pyridine

+ BSTFA (after 30 mins)

Determine δ13C of fructose and glucose by GC-IRMS

S. Gross & B. Glaser (2004) Rapid Communications in Mass SpectrometryVolume 18, Issue 22, 2753 –2764,


Ion Chromatogram of Derivatized Apple Juice

LC/EA-IRMS and GC-IRMS determination of δ13C of individualsugars in apple juices

23

24

25

26

27

28

1 2 3 4 5 6 7 8 9

10

δ 1

3C

CA carbohydrate

Polyamine

GC-IRMS

Fructose Glucose Fructose Glucose Fructose Glucose Fructose Glucose Fructose Glucose

KSAS 14043-02 KSAS 14520-02 KSAS 14788-03 KSAS 14280-03 KSAS 11249-04


Conclusions to Task 2.3:δ13C measurement on isolated sugars

• Fructose and glucose separated from apple, orange and pineapple juice on polyamine column have similar δ13C values

• A rapid GC/C/IRMS method has been developed for the confirmation of δ13C values of individual sugars in fruit juices

• The precision of the GC/C/IRMS measurements on individual sugars are comparable (~ 0.3 ‰ ) with the HPLC/ EA- IRMS measurements

• Consistent offset of 1.3 (+/-0.3) between δ13C of disaccharide and δ13C monosaccharide


T2.4 Determination of δ2H and δ13C in HMT derived from isolated sugars


•Periodic acid method•Quick chemical reaction•Can be used on many

organic molecules•Isotopic information

collected from specific sugar

•No isotopic ‘dilution’ of information by water

•Fermentation•1 week to ferment

sugars•Can only be used on

fermentable sugars•(D/H)I & (D/H)II

information ‘diluted’ by water

Periodic acid degradation vs fermentation


O

OHHO OH

CH2OH

OH

1

2

3

45

6

5H5IO6

5H5IO6

D-fructopyranose (60%)

4 HCOOH (C3 to C6, formic acid)CO2(C2, carbon dioxide)

H2CO(C1, formaldehyde)

2 H2CO(C1 and C6, formaldehyde)

CO2(C2, carbon dioxide)3 HCOOH (C3 to C5, formic acid)

(CH2)6N4

hexamethylene-tetramine(hexamethylenetetramine)

NH3

NH3

D-fructofuranose (40%)

mutarotation

O

CH2OH

OH

HO

H

HH

HOCH2

HO1

2

34

5

6

Fructose / Periodic acid Reaction


Formaldehyde conversion to HMT

Formaldehyde is reacted with ammonia to produce Hexamethylenetetramine (HMT). 6 HCH (CH2)6N4 + 4NH3 → O 6H2O


Quality control procedures

Sample

Semi-preparative HPLC

Amount of Fructose Recorded

Fructose Fraction Collected

Conversion to HMT

HMT purity determination using GC-FID

Weight of product Recorded

HMT Collected

HMT solution of known concentration

Amount of HMT Recorded,% yield Calculated


HMT n-hexadecane

Reference H 2 peaks

Time / seconds0 500 1000

5.6e-012

7.1e-009

4.2e-013

4.6e-011

m/z 2m/z 3

Ion Chromatogram


1100 oC

1200 oC

1300 oC

1400 oC

The Effect of Pyrolysis Temperature on HMT Peak Shape


δ 2H ‰

HMT STD1 (1200oC) -90.3HMT STD2 (1200oC) -88.9HMT STD3 (1200oC) -92.2HMT STD4 (1200oC) -87.7HMT STD5 (1200oC) -86.5HMT STD6 (1200oC) -88.8HMT STD7 (1200oC) -89.6HMT STD8 (1200oC) -91.5HMT STD9 (1200oC) -88.5HMT STD10 (1200oC) -87.5

Mean = -89.1SD = 1.7

δ 2H ‰

HMT STD1 (1400oC) -95.1HMT STD2 (1400oC) -102.0HMT STD3 (1400oC) -92.2HMT STD4 (1400oC) -94.3HMT STD5 (1400oC) -91.2HMT STD6 (1400oC) -87.7HMT STD7 (1400oC) -96.3HMT STD8 (1400oC) -95.2HMT STD9 (1400oC) -91.2HMT STD10 (1400oC) -93.0

Mean = -93.8SD = 3.9

1200oC

1400oC

The Effect of Pyrolysis Temperature on δ2H Reproducibility


Stage 1. Validation of HMT reaction procedure

Precision of δ 2H measurements of HMT derived fromstandard fructose

δ 2H ‰

S H 30 1 00 2a -2 36 .5S H 30 1 00 2a -2 35 .0S H 30 1 00 2a -2 32 .0S H 30 1 00 2b -2 41 .0S H 30 1 00 2b -2 40 .1S H 30 1 00 2b -2 38 .7S H 30 1 00 2c -2 35 .0S H 30 1 00 2c -2 32 .7S H 30 1 00 2c -2 35 .6S H 30 1 00 2d -2 39 .6S H 30 1 00 2d -2 39 .4S H 30 1 00 2d -2 38 .0S H 30 1 00 2e -2 33 .4S H 30 1 00 2e -2 33 .5S H 30 1 00 2e -2 36 .1S H 30 1 00 2 f -2 33 .2S H 30 1 00 2 f -2 36 .1S H 30 1 00 2 f -2 35 .2

Mean = -236.3SD = 2.2


Validation of fructose collection

GC-IRMS δ 2H measurements of HMT derived from fructosebefore and after HPLC using two different columns

Ca carbohydrate

-235.0

-238.0-233.5-236.7-231.5-234.9

2.6

Before Polyamine

1 -231.9 -238.42 -235.3 -232.83 -231.8 -236.44 -232.2 -234.75 -233.8 -229.0

Mean -233.0 -234.0SD 1.5 3.6


T2.6 Peer Testing of HMT method

28.7(3)

-29.1(3)

-77.8(3)

-36.7(3)

Lab5

-227.4 (4.2)

-52.2 (0.6)

26.0(2.9)

-28.3 (0.8)

-79.3 (3.3)

-36 (0.6)

Mean, (SD)

(GC-IRMS results)

-35.8 (5)

Lab4

10.0

-39.8

-93.2

-49.3

Lab3

-224.4 (3)

-52.6 (3)

-230.4 (5)

-51.8 (5)

HMT (fructose)

δ2H

δ13C

22.8 (5)

-27.6 (5)

26.4 (10)

-28.2 (10)

HMT (Fluka)

δ2H

δ13C

-83.1 (5)

-35.3 (5)

-76.9 (10),

-36.4(10)

HMT (Sigma)

δ2H (reps.)

δ13C (reps.)

Lab2Lab1Sample


δ 2H HMT

-200 -180 -160 -140 -120 -100 -80 -60 -40 -20

δ13

C H

MT

-40

-35

-30

-25

-20

-15

BrazilChinaCosta RicaFranceGermanyHondurasIndonesiaItalyKenyaMosambiquePhillipinesPolandRomaniaSpainThailand Turkey

δ2H and δ13C HMT results for Apple and Pineapple Juices

Apple Juices

Pineapple Juices

BMIS HFCSδ2H ~-250 ~-50δ13C ~-33 ~-17


Comparison of d2H values of HMT from apple juices organised by country

Tu

rke

y

Po

lan

d

Ch

ina

Ro

ma

nia

Ge

rma

ny

Ita

ly

Ukr

ain

e

Fra

nce

Sp

ain

δ2 H (

HM

T)

pe

r m

il

-200

-190

-180

-170

-160

-150

-140

-130


0 20 40 60 80 100

-240

-220

-200

-180

Effect of stepped addition of BMIS to Rumanian apple Juice sample

% BMIS

HM

T

2H

‰ V

-SM

OW

y = -65.769x - 166.19R2 = 0. 9865

-160

Values for Pure Apple juice


Country mean values

sample reference δ2H ‰ δ13C ‰

country of origin δ2H ‰ δ13C ‰ beet cane

AS01 -182.3 -30.2 Rumania -170(0.4) -33.45 (0.07) 10-30% 0AS02 -188 -31 Poland -174.3(6.4) -33.96 (1.59) 15-30% 0AS03 -181.5 -30 Rumania -170(0.4) -33.45 (0.07) 10-30% 0AS04 -180 -30.7 Germany -168.6(2.8) -33.96 (1.59) 10-30% 0AS05 -183.8 -30.1 Rumania -170(0.4) -33.45 (0.07) 15-35% 0

determined level of adulteration

Analysis of blind samples

T2.6 Validation

Range of D/H values for authentic juices -142 to-190 ‰


Country mean values actual

sample reference δ

2H ‰ δ13C ‰

country of origin δ

2H ‰ δ13C ‰ beet cane

AS01 -182.3 -30.2 Rumania -170(0.4) -33.45 (0.07) 10-30% 0 15%AS02 -188 -31 Poland -174.3(6.4) -33.96 (1.59) 15-30% 0 10%AS03 -181.5 -30 Rumania -170(0.4) -33.45 (0.07) 10-30% 0 5%AS04 -180 -30.7 Germany -168.6(2.8) -33.96 (1.59) 10-30% 0 0%AS05 -183.8 -30.1 Rumania -170(0.4) -33.45 (0.07) 15-35% 0 5%

determined level of adulteration

Analysis of blind samples


Comparison of d2H values of HMT from apple juices organised by country

Tu

rke

y

Po

lan

d

Ch

ina

Ro

ma

nia

Ge

rma

ny

Ita

ly

Ukr

ain

e

Fra

nce

Sp

ain

δ2 H (

HM

T)

pe

r m

il

-200

-190

-180

-170

-160

-150

-140

-130


Conclusions to Task 2.4

Determination of δ2H and δ13C in HMT derived from isolated sugars

• Optimisation of HMT pyrolysis and analysis by GC-PY-IRMS

• d2H values determined before and after HPLC show that there is no fractionation associated with the semi-prep chromatography

• Current detection limit approximately 26% for each country

• Analysis of 73 juices analysed for 13C and D/H of HMT now in database


• T2.5 Extension of HMT method to sorbitol from authentic apple samples as an internal isotopic reference

– Minor component unlikely to be adulterated, should give D/H values that can be related to the natural sugars

– Therefore potential internal reference for either SNIF NMR or

GC-PY-IRMS of HMT derivatives


Sorbitol / Periodic acid Reaction

Sorbitol Formic Acid (C2-5) Formaldehyde (C1-6) H O C1 HCOH C1 HCH C2 HCOH C2 HCOOH C3 HCOH C3 HCOOH

5 H5IO6 C4 HCOH C4 HCOOH → C5 HCOH C5 HCOOH C6 HCOH C6 HCH H O


Sorbitol Isolation

• Concentration of juice by 1/4 - 1/3

• Semi-prep LC fractionation of sugars

• 950µL injection volume


Time / minutes

0 10 20 30 40

RI

sig

na

l / m

V

0

20

40

60

80

apple juice after fermentation

apple juice before fermentation

FructoseGlucoseSucrose

Sorbitol

Effect of fermentation on the sugars in apple juice


Sorbitol method development

• Fermentation required large amounts of sample for sufficient sorbitol for HMT preparation (25-100 injections)

Before fermentation After fermentation

1 -223.5 -225.1

2 -224.6 -224.9

3 -226.5 -226.0

4 -225.6 -225.0

Mean -225.0 -225.25

SD 1.3 0.5

δ2H values of HMT prepared from sorbitol recovered from apple juice


δ2H HMT from fructose

-220 -210 -200 -190 -180 -170 -160 -150

δ2H

HM

T fro

m s

orb

itol

-220

-210

-200

-190

-180

-170

-160

-150

-140

EnglandGermanyPolandCzech RepublicAustriaUkraineNew ZealandFranceSouth Africa

Comparison of the δ2H values of HMT derived from sorbitoland fructose from 27 apple juices


(D/H1) / ppm

94 96 98 100 102 104 106

D/H

HM

T f

rom

so

rbito

l / p

pm

118

120

122

124

126

128

130

132

Comparison of the D/H ratio of HMT derived from sorbitol and the (D/H)1 ratio from SNIF-NMR analysis of 27 apple juices

EnglandGermanyPolandCzech RepublicAustriaUkraineNew ZealandFranceSouth Africa


Conclusions to Task 2.5Extension of HMT method to sorbitol from authentic apple samples as an internal isotopic reference

• Direct sorbitol collection has been exhaustively investigated and shown to be unrealistic because of the time required to collect sufficient quantity of sorbitol from apple juice

• Isolation of sorbitol from fermented apple juice has been performed however there was not found to be any significant isotopic correlation between the δ2H value of HMT derived from fructose or the (D/H)1 ratio of ethanol produced from the fermentation

• The isotopic analysis of sorbitol via HMT is therefore unlikely to improve the detection limit for beet sugar in apple juice


Summary of conclusions to WP2

• T2.2 Ion exchange purification of juice followed by Polyamine HPLC separation provides most reliable separation of individual sugars

• T2.3Fructose and glucose have similar δ13C values and there is a consistent offset of +1.3 (+/-0.3) between δ13C of sucrose and δ13C monosaccharide in apple, orange and pineapple juice

• T2.4Optimisation of HMT pyrolysis, confirmation of no fractionation during HPLC, optimisation of analysis by GC-PY-IRMS and analysis of 73 juices analysed for 13C and D/H of HMT now in database


Summary of conclusions to WP2

• T2.5 Concentrations of sorbitol in fruit juices are too low to be an economic internal reference and there is no correlation between δ2H value of HMT derived from fructose or the (D/H)1 ratio of ethanol produced from the fermentation

• T.2.6 Between laboratory confirmation of δ2H and δ13C values of HMT derived from fructose extracted from fruit juice


STAFF INVOLVED

• Dr Paul Brereton

• Dr Chris Rhodes

• Dr Simon Hird

• Janice Lofthouse

• Julie Christy

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