Transcript Aim of the presentation is to show you the powerpoint master

How will we be assessing fruit juices in the
future?
TCJJP meeting
Philadelphia 2009
Dr. David A Hammond
Eurofins Scientific
UK
E-mail: [email protected]
Fixed line 44 (0)118 935 4028
Mobile No. 44 (0)798 965 0953
www.eurofins.com
I really have no idea, so lets go down to the river
and have a beer!
2
What and why have we done in the past?
60’s
70’s
80 & 90’s
90’s into 00’s
Simple approach used single elements NPK
(potassium, nitrogen and phosphorous)
General approach developed which included minerals,
organic acids, amino acids, sugars, 13C-SIRA applied to
fruit juices
Multi-component approaches (RSK, AFNOR)
Isotopic methods developed, trace minerals (country of
origin)
“AIJN COP”
Internal isotopic relationships developed
fingerprinting methods, heavy elemental isotopic analysis
(87Sr), DNA fingerprinting methods
3
Why have we developed?
60’s
It soon became clear that by just looking at potassium, nitrogen
and phosphorous this was not enough
70’s
profile of free amino acids were no longer enough had to
move onto look at D,L-amino acids
80’s
Methods based solely on conventional parameters (“RSK”
approach) was no longer sensitive enough & needed isotopic
methods
90’s
Isotopic methods needed refinement to enhance their
sensitivity
00’s
Newer methods needed to look at specific problems “Pure
Juice Project”, DNA fingerprinting methods, Screening methods
1H-NMR (more “cost effective” screening)
4
New approaches adopted for three major reasons
A new technology became available that was better/quicker/easier
 LC sugars
 LC for flavanoids
Problem identified with a specific technology
 SNIF-NMR® was developed to target the detection of addition of
beet sugars to wine must (grape juice) later applied to fruit
juices, honey, maple syrup
 Oligosaccharide screening by HPAEC-PAD marker
trisaccharides in sugar syrups
 DNA screening for mandarin in orange juice
Old technology being used in a new area
 Serendipity “look see” with new application HIS by GC-FID
5
Enhancement of cane/corn addition
How can we limit addition of cane/corn sugar addition?
Suggested in mid 70’s to use a internal reference (pulp)
Not all juices contain pulp to use (apple)
Are there other materials that can be used?
Isotope ratios seen in sugars and acids and between
individual sugars and acids
6
Enhancement of 13C methodology
Centrifuge
Fruit Juice
Separation
of
Fractions
Neutral
H PLC S e p a ra t i o n
Of S U G A R S
Carbon-13 determination of
Whole juice - Sucrose Glucose - Fructose
Isolation of
Soluble Pectin
Acid
H PLC S e p a ra t i o n
of A C I D S
Carbon-13 determination
of
Citric - Malic Acids
Carbon-13
determination
7
Control addition of cane sucrose to orange juice
-20
CS8%vol
-21
CS4%vol
-22
δ 13C
sucrose
CS2%vol
-23
-24
Reference data (ALL)
Starting juice
-25
-26
Adulterated juices
-27
-26
δ
-25
13
-24
-23
C glucose
8
Enhancement of 13C methodology
Centrifuge
Fruit Juice
Separation
of
Fractions
Neutral
H PLC S e p a ra t i o n
Of S U G A R S
Carbon-13 determination of
Whole juice - Sucrose Glucose - Fructose
Isolation of
Soluble Pectin
Acid
H PLC S e p a ra t i o n
of A C I D S
Carbon-13 determination
of
Citric - Malic Acids
Carbon-13
determination
9
Enhancement of acid/sugar addition intercompound detection
10
Inter-molecular 13C measurement to enhance C4
sugar/acid addition
1.
13C
2.
13C
ratios in individual sugars can drop the detection limit to ca
5% where tailor made blends of beet & cane/corn sugars are used
ratios of citric and malic and total sugars can enhance the
addition of C4 derived materials down to ca 10%
11
Intra-molecular bi-isotope ratio testing to enhance
detection of citric acid addition to juices
Here decided to look at two different isotopes within the same molecule
Examined overall
addition
13C
and bound hydrogen D/H ratios to look for exogenous citric acid
CO2H
|
CH2
|
HO-C-CO2H
|
CH2
|
CO2H
Entailed isolation of citric acid from juice by precipitation as its calcium salt
Analysis of the δ13C by IRMS and D/H ratio in non-exchangeable hydrogen by
pyrolysis-IRMS, which was much simpler that the previous procedure by SNIF-NMR®
12
Intra-isotope ratio testing to enhance detection of
citric acid addition to juices
13
Detection of citric acid addition to commercial
lemon juices
14
SNIF-NMR®
13C
& D isotope ratio mass
spectrometry used to detect
sugar addition
13C
-10
-12
The latter was difficult to
apply in practice
-14
-16
SNIF-NMR simplified beet
sugar detection
-18
-20
More problems occurred
where (D/H)I & δ13C were
suspect!
-22
-24
Required a new development
(‰)
-26
-28
90
95
100
105
110
115
D/H1 (ppm)
15
Fingerprinting methods
 Anthocyanin profile (this will be used in the future as it is central
to the assessment of red/black juices)
 Oligosaccharide profile (I think this will still be used and there
may well be more developments in this area either using new
markers for new syrups, or new detection methods)
 Polyphenol profile (I think that this approach will be used more in
the future)
16
Anthocyanin profile for a juice adulteration
Pattern of anthocyanin peaks is often characteristic of a fruit type
Critical approach to use when examining red/black fruit
Methodology validated by IFU analytical Commission
Available for a fee from the IFU website 500 euros for ca 50 chemical
method, 200euros for 12 micro methods designed for fruit juices or
650 euros for both sets. Downloadable via credit card payment.
www.IFU-fruitjuice.com
17
Blackcurrant anthocyanin profile
Delphinidin-3-rutinoside
Cyanidin-3-glucoside
10.359
mAU
500
9.357
DAD1 A, Sig=520,4 Ref=590,20 (ANTH0603\18774-1.D)
400
300
100
Cyanidin-3-rutinoside
10.148
Delphinidin-3-glucoside
9.118
200
0
2
4
6
8
10
12
14
16
18
min
18
Cranberry anthocyanin profile
DAD1 A, Sig=520,4 Ref=590,20 (ANT H1403\MACRA.D)
mAU
Peonidin-3-galactoside
Cyanidin-3-galactoside
200
Peonidin-3-glucoside
150
Cyanidin-3-arabinoside
100
Peonidin-3-arabinoside
50
0
2
4
6
8
10
12
14
16
18
min
mAU
Peak too strong
relative to other
anthocyanins for
cranberry
Additional peaks
40
30
Elderberry addition
20
10
0
2
4
6
8
10
12
14
16
18
min
19
Polyphenol profiles
Again can be characteristic of fruit or even fruit variety!
Often very complex pattern of peaks (“forest”)
Peak retention time is a critical element (too variable to be sure of
peak assignment in some cases)
Can be very useful in some simple cases (grapefruit in orange, sour
orange in sweet orange)
Not a validated method as yet!
20
Simple polyphenol issue grapefruit in orange
100
Chroma togram
12
Hesperidin
Narirutin
8
50
mVolts
% Mobile Phase
10
6
4
2
0
0
-2
0
10
20
30
40
Minute s
c:\gilson\rssl\ system_ 1\po lyp he.0 01\po lyphe.g dt : UV ch anne l 1 : oj: Inj. Num ber: 2
100
12
8
50
mVolts
% Mobile Phase
10
Naringin
6
4
2
0
0
-2
0
10
20
30
40
Minute s
c:\gilson\rssl\ system_ 1\po lyp he.0 01\po lyphe.g dt : UV ch anne l 1 : oj/re ef: Inj. Numbe r: 4
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Difficult polyphenol issue
Pineapple skin extract addition to juice
Skin related peaks
100
50
mVolts
% Mobile Phase
10
0
0
10
20
30
Minutes
c:\gilson\rssl\system_1\polyphen.017\polyphen.gdt : UV channel 1 : 10535 : Inj. Number: 7
c:\gilson\rssl\system_1\polyphen.017\polyphen.gdt : UV channel 1 : 10535 5%mexico: Inj. Number: 8
c:\gilson\rssl\system_1\polyphen.017\polyphen.gdt : UV channel 1 : 10535 10%mexico: Inj. Number: 9
c:\gilson\rssl\system_1\polyphen.017\polyphen.gdt : UV channel 1 : 10535 20%mexico: Inj. Number: 10
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Aroma profiling in juices
 Very complex area
 Specialist analysis
 Many compounds of interest
Detects addition of synthetic flavours
 Presence of solvent/flavour carriers
 Chiral GC
 GC-IRMS
This is an approach that will be used in Europe where there will
pressure to prove pure FTNF aromas are being used.
23
Detection of Mandarin in Orange
“New” DNA methods

LFRA mandarin method developed for UK Food Standards (mid
90’s & 08 with Lab on Chip method Hetero-duplex procedure)


AZTI method developed in Spain
French method IDNA for mandarin and mango
24
Gel showing two bands for orange/mandarin blends
Original LFRA hetero-duplex method
O
1
10
50 90 %
O
1
10
50
90 %
O
1
25
50
90 %
M
25
Lab on a chip DNA screening RFLP
26
ADNid microsatellites SSR
27
More rapid screening methods will be investigated
In the past FT-IR, NIR and pyrolysis-mass spectrometry, coupled with
using multivariate statistical techniques have been proposed as
rapid screening methods. However, none of these have stood the
passage of time
CSL in York have done work in this area on a range of food products
& SGF & Eurofins are looking at using 1H-NMR to fingerprint juices at
present.
The method involves the collect a simple NMR spectrum and use
multivariate statistical methods to determine if the sample is
“normal” or not (better targeting of the “analysis euro”).
28
1H-NMR
profiling
Fast & simple =
Composition in 15min, with limited sample preparation
Qualitative =
Detection or absence of a specific characteristic
compound or tracer
Quantitative =
Concentrations can be determined with accuracy & precision
Comparable to chromatography
Powerful discrimination =
Statistical treatment of the signal
= « profiling » approach
29
Comparison of 1H-NMR and IFU sugar methods
Conclusion:
The 1H- NMR measurement
of concentrations is accurate
30
1H-NMR
profiling
 Principle : even the small signals from
the spectra provide authenticity
information
 Statistical comparison of the 1H-NMR
spectra of the juice with the database of
authentic product
 Discrimination:
- Typical / atypical juice
- Orange / Reticulata (addition)
- FC / NFC
…
Red = Orange
Blue = Reticulata
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1H-NMR
profiling for NFC vs FC products
FC
NFC
fc 02092071101
nfc12191151101
fc 02231921101
fc 02231921 b101
fc 02231923101
PLS-DA Pareto 68fc 123nfc
0.1
nfc02224496-2101
fc 02231931101
fc 02231940101
nfc02142613101
fc 02231935101
nfc02231933
nfc02196508
0 0
1 1 1 1
nfc02231934101
fc 02192461 0
fc 02142610101 1 1
fc 02231922101
t2
nfc02187992101
fc 02142618101
fc 02192156101
fc 02172228101
fc 02142621
0
nfc02198332
nfc02172224110
011
1 1
fc 02197156
01
nfc02172530nfc02192349
0
1 1
0
fc 02172226
0
nfc02092065101
fc 1
02193828
0
fc 02172229
002231939
1 1
1 1
1 1 fc
0
1 1
1 1
nfc02172221101
nfc02195798101
fc 02186588
0
fc 02142644
0
nfc02171032101
1 1
nfc02197374101
1 1
nfc02224496
0.05
0
fc
02166588
fc
02183879
0
0
fc
02021966
0
nfc02194240
0
1 1
1 1
1 1
1 1
1 1
fc 02191396
0
1 1
nfc02142650
0
nfc02142658
0
fc 02172231 0
0
nfc02231926
0 nfc02173930
fc 021426191fc
01 02231937101
11 110
nfc02231925
nfc02231927
fc 021426081011fc1 02231930
1 1
1 1
nfc02198448101
1 1 101
01
fc 02171610
0
nfc02197376 0
1 0
fc 02187116
0102196109
1fc1 02197174
fc 02196447
01
1
fc
0fc
1 02142643
nfc02190064101 1 1
1 1
0
1
1
nfc02142638101
1 1
fc 02163921101
nfc02172220101
nfc02194252
0
nfc02194241
0
1 1
fc 02196805101
1 1
nfc02231928
01
nfc02142633
01
1
1
nfc02170963
0
fc 02194120101
nfc02197168
0
fc 02142606
1 1
1 10 0
fc 02142635
0 101 fc 02199165
nfc02173786
nfc02180824
0
1 1
nfc02172215
1 011 1
nfc02181019
fc
fc
fc02170604
02198743
02183874
0
0
01 1 1
1 1 101
11
1 11
nfc02193091101
fc
02194086
0
nfc02190282
nfc02191506110
011
1 1
nfc02216791101
fcfc02194236
01 1
02021962
nfc02142607nfc02175170
0
nfc02194247
0
nfc02142648
0
1 10
1 1
1 10
nfc12191572
0
101
1
fc 02196983101
fc 02231936101
1
nfc12191180
1 1 nfc02191919
0101
nfc02142656
0
nfc02142637
0
nfc02204827
1 1
1 1
1 1
nfc02198822
0 101
021678140101
nfc02205712
01
fcfc
02194986
1 1
1
1
1
nfc02212760
01 nfc02231924 0
nfc02142620
0
fc 02204825
0
nfc02197377
fc 02211957
10
1 1
1 1 101
nfc02200062
0 1 1
10
1
nfc02171011
0
fc 02198432
0
nfc02142634
1 1nfc02192157
nfc02205714
0 0
1 1 nfc02170773
1 1
0
fc 02211521
0 fc 02115386101
1 1
1 11 1
1 1
1 1
fc 02199143
0
1 1
nfc02205715101
nfc02051513
0
nfc02051501
0
nfc02171448
0
1 1
nfc02198684101
nfc02198456
0
nfc02198302
0
1 1
1 1
1 1 0
1 1
nfc02191013
1nfc02194437
1
nfc02172214
01 1010nfc02194461
0
nfc12191155101
nfc12191569
0
0
nfc02224497-2
1 nfc02175095
1 1
1 1
1 1
nfc12191176
0
1 1
1 1
nfc02224497
0
nfc02186593
0
1 1
1 1
nfc02142645101nfc02211518
0
fc 02192460101
1 1
nfc02196809101
-0.05
nfc02196808
nfc02114452
01
nfc02051498
0
1
1 1
nfc02197466101
1 1
nfc02192393101
nfc02196781101
fc 02142653101
nfc02142660101
fc 02186824101
nfc02206449101
nfc02051503
0
nfc02211527
0
1 1
1 1
nfc02212759101
-0.1
nfc02062372101
nfc02062381101
fc 02142651101
nfc02114242101
nfc02151610101
nfc02142646101
nfc02107327101
nfc02142641
0
nfc02062394
0
1 1
1 1
-0.1
-0.05
0
fc 02140683
fc1002142659
0
1
1 1
nfc02231938101
0.05
nfc02142630101
0.1
0.15
t1
fc 02142640101
nfc02142654101
nfc02021950101
nfc12191153101
nfc02205713101
nfc02021946101
nfc02171803101
nfc02181724101
32
Points to remember!
Most suppliers are honest but we need to weed out those that are
not!
Test as much as you can afford!
Use an expert laboratory who is ISO 17025 approved!
If you are testing for yourself use methods that have been validated,
such as IFU or AOAC, and confirm your performance is within line
with the data published for the method (repeatability and
reproducibility).
Run quality control samples to monitor drift and analyst performance
Subscribe to a laboratory check sample program
33
What Next?
1. Further development of internal isotopic methods to enhance detection limits are
likely.
2. DNA methodologies are likely to become used more, although juices do not offer
an ideal medium for their use, due to damage to plant DNA by heat and acid during
processing.
3. Revitalisation of rapid screening methods 1H-NMR
4. Maybe new developments in the oligosaccharide screening area
5. Country of origin maybe come an issue with “carbon miles” and peoples’ desire to
know more about their food. Here trace minerals such as Rb, Ba, Sr will be of
interest as will 87Sr analysis.
6. Analysis of functional ingredients in juices, both for quality and authenticity needs
will become an important issue.
7. I am sure that there will be new techniques used that are not presently applied in
the FJ area at present. LC-MS is likely to be more commonly used in the future
8. Lab accrediation and use of validated methods are likely to be important
34
Points to remember!
Use materials from an approved list and visit/audit their factories. Or
use product produced by a IRMA approved supplier as the audits
have already been done for you by experts!
Be more vigilant when prices are high, as a number of “skeletons
often come out the cupboard” then!
It is much easier to loose a good reputation than it is to develop one!
Don’t forget some good deals are just too good to be true and the
buyer should always beware!!!!
Thank you for your attention
35