lc-ms/ms analysis of marker compounds for sensory quality ...€¦ · 2.0 0.1 13.2 20.7 14.8...
TRANSCRIPT
© 2016 Technische Universität München
LC-MS/MS analysis of marker
compounds for sensory quality
and authenticity of fruit juices
Andreas Dunkel
SCIEX Innovation Day, Analytica, 160511
© 2016 Technische Universität München
simplified sample prep.
inexpensive
high robustness
high reproducibility
library creation
automation possible
combinatorial odor/taste
codes required
instrumental
Mass spectrometry
(SENSOMICS)
direct sample analysis
expensive & time consuming
subjective vs. objective
sensory fatigue
volatility of experience
personnel fluctuation
skilled personnel required
in vivo
Human sensory analysis
(trained panelists)
massive sample prep.
multiple assays needed
low robustness
prone to interferences
library creation
crowded IP landscape
skilled personnel required
in vitro
Cell-based or bioelectronic
taste receptor assays
Tools for aroma/taste assessment
© 2016 Technische Universität München
Direct juice (DJ) vs. juice from concentrate (CJ)
• European market: predominantly apple
and orange juices from concentrate
• Insufficient reconstitution affects the quality of
CJ, consumer protection organizations
increasingly report a decline in sensory quality
• Technological challenge to achieve organoleptic/
analytical equivalence of DJ and CJ as required
by legal regulations (Fruchtsaftverordnung)
• Current situation: only verification of the
„analytical similarity“ insufficient verification
of the sensory quality of the reconstituted juice
• Aims: improvement of the sensory quality of
apple and orange juices from concentrate,
quantification of the crucial sensory active
compounds, development of objective parameters
for evaluation of sensory quality and simplified
analytical methods
© 2016 Technische Universität München
Which molecules hit our receptor repertoire during food consumption?
© 2016 Technische Universität München
HILIC-MS/MS(ESI+)-SIDA Tosoh TSK-gel Amide 80,
150 x 2.0 mm, 3 µm
ZIC-pHILIC-MS/MS(ESI-)-SIDA Sequant polymeric ZIC-pHILIC
150 x 4.6 mm i.d., 5 µm
LC-MS/MS-SIDA quantitation of “generalist“ tastants
RP18-HPLC MS/MS(ESI-)-SIDA Phenomenex Synergi Hydro-RP
4µm, 80Å, 150 x 2.0 mm
© 2016 Technische Universität München
Isolation of sweet taste modulating molecules from apples
• Recombinant of basic tastants from apple juice shows an non-characteristic sweet-sour profile
Prep. RP18-HPLC – Taste Dilution Analysis
EtOAc extract induces
change in sweet/sour
profile (α < 0.05)
Fraction VIII of EtOAc
extract induces change in
sweet/sour profile (α < 0.05)
Prep. PheHex-HPLC of subfraction VIII
© 2016 Technische Universität München
Structures and taste thresholds of ursane triterpenoids
H3C(25)
HC(1)
H3C(29)
HC(18)
HC(20)
ROESY, 500 MHz, d3-MeOH
euscaphic acid
(25 µmol/L)
corosolic acid
(13.2 µmol/L)
ursolic acid
(14.8 µmol/L)
annurconic acid
(20.7 µmol/L)
annurcoic acid
(0.1 µmol/L)
• Structure elucidation of sweet taste modulating ursane
triterpenoids by means of 1D/2D-NMR and MS experiments
© 2016 Technische Universität München
LC-MS/MSMRM quantitation of ursane triterpenoids
0 4 8 12 16 20 24 28 time [min]
Inte
nsity, cp
s
recovery expt. in recombinant
compound recovery [%]
euscaphic acid 97.6 ± 0.3
annurcoic acid 100.58 ± 0.04
corosolic acid 108.7 ± 2.5
annurconic acid 105.7 ± 0.8
ursolic acid 108.9 ± 1.2
5500 QTrap
Ekspert microLC 200
• Application of micro-UHPLC enables direct quantitation without sample workup
Stationary phase: Sciex ChromXP C18-AQ (3µm, 150 mm x 0.3 mm, 120Å)
Mobile phase: H2O/ACN (1%FA each)
Sample volume: 1 µL
© 2016 Technische Universität München
Annurcoic acid
Corosolic acid
Annurconic acid
Ursolic acid
Euscaphic acid
1.9
0.6
0.6
0.5
2.0
0.1
13.2
20.7
14.8
>25.0
21.0
< 0.1
< 0.1
< 0.1
<0.1
LC-MS/MSMRM quantitation of ursane triterpenoids
0 4 8 12 16 20 24 28 time [min]
Inte
nsity, cp
s
recovery expt. in recombinant
compound recovery [%]
euscaphic acid 97.6 ± 0.3
annurcoic acid 100.58 ± 0.04
corosolic acid 108.7 ± 2.5
annurconic acid 105.7 ± 0.8
ursolic acid 108.9 ± 1.2
5500 QTrap
Ekspert microLC 200
sweet
modulating
Tastant Conc [µmol/L] Threshold [µmol/L] DoT
• Application of micro-UHPLC enables direct quantitation without sample workup
© 2016 Technische Universität München
aspartic acid K+ phlorizin quercitrin annurcoic acid
Direct juice (DJ) vs. juice from concentrate (CJ)
Direct juice
Juice from concentrate
© 2016 Technische Universität München
Taste active compounds in orange juice
minerals polyphenols carbohydrates organic acids amino acids
limonoids and
limonoid glucosides
polymethoxylated
flavones
O
O
O
O
O
O
O
O
Limonin
Glabasnia (2008)
9 µmol/L (bitter)
19 µmol/L (astr.)
3,5,6,7,3´,4´-HMF
150 µmol/L (bitter)
31 µmol/L (astr.)
5,6,7,4´-TMF Nomilin-17-β-D-glcp
4 µmol/L (bitter) > 700 µmol/L (bitter)
© 2016 Technische Universität München
Quantitation method for the bitter principles of orange juices
• 2 Limonoids, 6 limonoid glycosides, and 9 polymethoxylated flavones could be detected in orange
juice samples as well as process steps
• Commercially available internal standards (e.g. 3,5,7,3‘,4‘-pentamethoxyflavone for PMF‘s)
• Direct injection of samples after filtration)
t [min]
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25
Micro-HPLC-MS/MS: Sciex QTRAP 5500 + Ekspert 200
Stationary phase: Phenomenex Synergi Fusion-RP (4µm, 50 mm x 0.3 mm, 80Å)
Mobile phase: H2O/ACN (0.1%FA each)
Sample volume: 1 µL
Multivariate analysis of commercial orange juices (german market)
© 2016 Technische Universität München
−4
0
4
8
−10 −5 0 5Comp.1
Com
p.2
• Score plot Partial least squares – discriminant analyisis
(non-linear iterative partial least squares algorithm, leave-one-out cross validation,
unit variance scaled
Direct juice (cold storage)
Direct juice
Juice from concentrate
© 2012 Technische Universität München
Modeling of orange juice bitter taste
sugarsterpenesPMFsterpenesPMFs *Bitter taste
intensity =
Model experiments
Variation of sugar content Variation of sugar content
Va
riation o
f P
MF
conte
nt
Varia
tion o
f lim
onoid
conte
nt
© 2012 Technische Universität München
Modeling of orange juice bitter taste – „Calibration“
56
78
9
10
11
0
1
2
3
4
5
6
7
1,4
1,2
1,00,8
0,60,4
0,2
Correlation between bitterness and concentration of sugars (PMFs konst. 1.1 mg/100 g)
bitte
r in
tensity
Concentra
tion o
f Lim
onoids [m
g/100 g
]
Concentration of sugars [g/100 g]
0
0,8750
1,750
2,625
3,500
4,375
5,250
6,125
7,000
© 2012 Technische Universität München
Modeling of orange juice bitter taste
0 1 2 3 4 5
0
1
2
3
4
5
estimated bitter taste intensity
bitte
r ta
ste
inte
nsity
Calibration
R2 = 0.98
Commercial Juice Samples
0
1
2
3
4
5
0 1 2 3 4 5
bitte
r ta
ste
inte
nsity
estimated bitter taste intensity
© 2016 Technische Universität München
Pushing limits in controlling “food quality“ based on profound
scientific knowledge
No food quality without mass spectrometry!
© 2014 Technische Universität München
Many thanks!
© 2016 Technische Universität München