Download Analysis of 16 amino acids in tobacco by ion pair

Analysis of 16 amino acids in tobacco by ion pair LC
ESI MS MS without derivatization
M. Bouzige, L. Barbet, V. Ramon,
F. Bourcier, B. Vidal, B. Duméry
ALTADIS
France
Introduction
Amino acids (AA) are chemical markers of tobacco type, maturity,
origin...
Two drawbacks for their analysis : lack of chromophoric agents and
polarity
Derivation before their detection by UV or fluorescence : This step
is often time consuming and can lead to extra errors
No derivation by using ionic exchange or ion pair chromatography :
problems of specificity
Mass spectrometry can overcome this problem
TSRC 2003 - Norfolk
LC MS MS analysis of Amino Acids
Work of the Organic and Analytic Chemistry Institute of
Orléans University (ICOA)
Determination of 20 underivatized proteinic amino acids by ion
pairing chromatography and pneumatically assisted electrospray
mass spectrometry.
• P Chaimbault, K. Petritis, C. Elfakir, M. Dreux.
• J. Chromatogr. A 855 (1999) 191 - 202.
Parameter optimization for the analysis of underivatized protein
amino acids by liquid chromatography and ionspray tandem mass
spectrometry.
• K. Petritis, P. Chaimbault, C. Elfakir, M. Dreux.
• J. Chromatogr. A 896 (2000) 253 - 263.
TSRC 2003 - Norfolk
Purpose
Adapt the method to tobacco
Extraction
Separation
Ion pair HPLC
Detection
ESI MS MS
Study its feasibility, performance
Compare its results with our traditional method
This method allows to analyze minor amino acids we
don’t currently analyze
Study the interest of these new amino acids for the
characterization of tobaccos
TSRC 2003 - Norfolk
MS MS Conditions
Apparatus : Perkin Elmer Sciex
API 300 triple quadrupole
Ionization : electrospray, positive
mode
Curtain and collision gas : Nitrogen
According to the publications, we
verified
MS parameters for ionization
Fragmentation products for
each AA
Best collision energy suitable
for all the AA : 20 eV
Scan mode : Multiple Reaction
Monitoring
Compounds
Alanine (ALA)
Arginine (ARG)
Asparagine (ASN)
Aspartic Acid (ASP)
Glutamic Acid (GLU)
Glutamine (GLN)
Lysine (LYS)
Histidine (HIS)
Leucine (LEU)
Isoleucine (ILE)
Methionine (MET)
Phenylalanine (PHE)
Proline (PRO)
Serine (SER)
Threonine (THR)
Tryptophane (TRP)
Tyrosine (TYR)
Valine (VAL)
Q1/Q3
90/44
175/175
133/87
134/88
148/130
147/130
156/110
132/86
150/104
166/120
116/70
106/60
120/74
205/188
182/165
118/72
TSRC 2003 - Norfolk
Amino acids
H 2N
CH 2 CONH 2
H2N
CH
COOH
Asparagine (ASN)
133 / 87
CH2CH2COOH
CH
CH3
Aspartic acid (ASP)
134 / 88
H2N
COOH
CH
CH
COOH
H2N
NH2
CH2COOH
Alanine (ALA)
90 / 44
CH2CH2CONH2
CH
COOH
N
H
COOH
Glutamic Ac. (GLU)
148 / 130
Glutamine (GLN)
147 / 130
COOH
Proline (PRO)
116 / 70
NH
H2N
COOH
CH2OH
CH
COOH
H2N
NH
CH
N
H
2
COOH
Serine (SER)
106 / 60
Phenylalanine (PHE)
166 / 120
Arginine (ARG)
175 / 175
TSRC 2003 - Norfolk
NH2
Amino acids (2)
H2N
CH(CH3)2
CH
H2
C
H N
2
H2N
HCH2CH3
C
CH
COOH
COOH
Valine (VAL)
118 / 72
NH2
CH
N
CH
COOH
H2N
N
Histidine (HIS)
156 / 110
H2N
CH2CH(CH3)2
CH
CH3
Isoleucine (ILE)
132 / 86
H2N
CH
COOH
COOH
Lysine (LYS)
147 / 130
Leucine (LEU)
132 / 86
N
H
COOH
Tryptophane (TRP)
205 / 188
OH
NH2
COOH
C
H2
H2N
CH2CH2SCH3
CH
COOH
CH
H2N
CH
CH3
COOH
Glycine (GLY)
76 / 30
Methionine (MET)
150 / 104
Threonine (THR)
120 / 74
TSRC 2003 - Norfolk
LC Conditions
Column : C18 silica. Lichrocart (Purospher) 125 x 2 mm, 5 µm
Mobile phase :
A = Heptafluorobutyric acid (HFBA) 0.1 % / ACN 97.5 / 2.5
B = ACN
Flow rate : 0.2 mL/min, split 1/10 before ion source
Gradient elution
120
100
100
Necessity to add a
washing step with pure
ACN between two runs
% B
80
60
40
30
30
Equilibrating time 30 min
20
0
0
5
10
15
20
T (min)
25
30
35
TSRC 2003 - Norfolk
Sample preparation
Standards
Tobacco
Necessity to add HFBA
in the sample to improve
the formation of ion pair
of HIS, ARG, LYS at the
injection
Extraction
1 g of tobacco powder in 50 mL of
water
Internal Standard :
choice of AA not
detected in a series of
tobaccos : methionine &
threonine
Dilution of the extract by 2
Addition of HFBA : 0.3 % of HFBA
are necessary to obtain symmetric
peaks for HIS, ARG, LYS.
Methionine chosen as Internal
Standard. Threonine was rejected
due to matrix effect on tobacco.
TSRC 2003 - Norfolk
Analysis of a Burley tobacco
Superposition of the 18 SRM
Nicotine
PRO
PHE
ASP
ASN
MET
GLN
SER
ALA
GLU
HIS
VAL
LYS
TRP
ILE
LEU
ARG
TYR
Time
min
Specificity of MS MS allows to decrease the separation
TSRC 2003 - Norfolk
Matrix Effect
Tobacco
(Burley, FlueCured, Oriental)
FC
Tryptophane (TRP)
Int, Ratio
1,6
Filetype
FC
Standard
1,2
5 spikes
3 repetitions
2
Linearity of the response
3
Comparison with standard
4
Recovery calculation
0,8
0,4
0
0
1
2
3
4
Amino acid concentration /100
(mg/kg of dry tobacco)
Blank
1
Linearity of standards
5 concentrations
3 repetitions
TSRC 2003 - Norfolk
Recoveries
AA
ASN
SER
ASP
GLU
GLN
ALA
PRO
HIS
LYS
VAL
ARG
ILE
LEU
PHE
TRP
TYR
Burley
Flue Cured
Oriental
Recovery (%) Recovery (%) Recovery (%)
98
106
109
87
102
104
103
103
105
99
84
93
98
103
103
94
102
106
97
102
107
108
110
103
103
98
98
81
81
82
102
95
94
89
103
100
86
100
97
100
109
102
105
103
102
86
82
95
Linearity of standards and spiked tobaccos always
validated
Some matrix effects but acceptable
TSRC 2003 - Norfolk
Repeatability & Limit of quantification
Analysis of a Burley tobacco
during 6 weeks over 4 months
AA
ALA
ARG
ASN
ASP
GLN
GLU
HIS
ILE
LEU
LYS
PHE
PRO
SER
TRP
TYR
VAL
CV %
7
15
13
10
18
13
15
9
14
17
15
13
11
13
20
14
Estimation of LOQ, comparison with
the mean value observed after
analysis of 106 various tobaccos
Necessity to calibrate
every 3 days
Deuterated Internal
Standards should
improve the
repeatability
Limits of
quantification
compatible with the
analysis of tobaccos
LOQ
mg/Kg
6
3
13
3
3
25
4
10
2
3
0.3
2
4
0.5
4
2
Mean
411
149
5424
5443
713
561
271
40
49
192
464
3572
275
276
71
82
TSRC 2003 - Norfolk
Comparison with our current method
New Method
Current Method
Extraction
Extraction
Ion pair HPLC
ESI MS MS
Analysis of tobaccos
Derivation
28 Burleys
38 Flue Cured
13 Oriental
Reverse phase HPLC
UV or Fluorimetry
Asn,Asp, Glu,
Gln, Ser, Pro
Asn,Asp, Glu,
Gln, Ser, Pro
Ala, Arg, His,
Tyr, Val, Leu,
Ile, Trp, Phe,
Lys
Ala, Arg, His
TSRC 2003 - Norfolk
Comparison with our current method
AA
R2 adjusted
Global By
FC
O
ASN
91.6
91
81
99
SER
77
84
51
96
ASP
96.6
84
84
99
GLN
80
80
67
75
GLU
79
95
9
93
PRO
91
95
77 64.6
ARG
HIS
ALA
95
90.9
16.6
92
94
62
58
78
3
18000
Slope
Global
1.03
1.13
1.16
1.02
0.94
0.88
96
94
32
1.16
1.05
0.3
1400
Comparison of ASP results
16000
Comparison of HIS results
1200
14000
1000
ESI LC MS MS
ESI LC MS MS
y = 1.1608x + 10.45
12000
2
R = 0.9667
10000
8000
6000
800
600
y = 1.0524x - 1.3003
400
2
R = 0.9094
4000
200
2000
0
0
200
400
600
800
1000
0
0
2000
4000
6000
8000
10000
Current method
12000
14000
16000
-200
Current method
TSRC 2003 - Norfolk
1200
Comparison with our current method
Similar results
Shorter analysis time
no derivation
faster separation
Lower limits of detection for amino acids : at least a factor 10
Possibility of analyzing new amino acids
Reproducibility should be improved
TSRC 2003 - Norfolk
Application to the characterization of tobaccos
Extraction
Separation
Ion pair HPLC
Detection
ESI MS MS
Analysis of tobaccos
28 Burleys
27 Dark
38 Flue Cured
13 Oriental
New AA compared
to our current
method
Do these new AA improve
the characterization of
tobaccos ?
TSRC 2003 - Norfolk
Differences between tobacco types
Box and Whiskers
Box and Whiskers
BRU
TYPE
BUR
ORI
VIR
BUR
ORI
VIR
0
300
600
900
1200
0
1500
30
HIS
60
90
120
150
ILE
900
Mean DARK
Mean BURLEY
Mean ORIENTAL
Mean FC
800
Concentration in mg/kg of dry tobacco
TYPE
BRU
700
600
500
400
300
200
100
0
HIS
TRP
PHE
ARG
VAL
LYS
TYR
ILE
LEU
TSRC 2003 - Norfolk
Selection of the best amino acids
5.30
Component 2 - Inertia: 23.9%
BRU
Factorial plane
Correlation circle
ASP
3.10
BRU
BRU
0.90
-1.30
BRU BRU
BRU
BRU
BRUBRU BRU
BY
BRU
BRU
BRU
BRU BRUBY
BRU
BRU
BRU
BRU
BY
BRU BY
BRU
BY
BRU
BY
BY
BRU
BY
VIR
VIRVIRVIRVIR
VIR
VIR
VIR
VIR VIR
VIRVIR
VIR
VIR
VIR
VIR
VIR
VIR
VI
VIR
VIR VIR
ORI VIR VIRVIR
VI
VIRR VIR
VIR
R
VIR ORIVIR
VIR
VIR
ORI ORI
VIR VIR
VIR VIR
ORI
OR ORI
ORI
ORI
I
VIR
LYS
BRU
BY
BY
BRU
BY
BY
BY BY BY
BY BY BY
BY
BY
BY
VAL
ASN
BY
BY BY
BY
BY
TRP
HIS
ALA
BY
BY
ORI
PRO
ORI
ORI ORI
-3.50
-3.00
-0.50
2.00
4.50
7.00
Component 1 - Inertia: 53.8%
Ala, Trp, His, Val, Lys bring some information
AA separate well Burley tobaccos
TSRC 2003 - Norfolk
Conclusion
Amino acids can be analyzed by ESI LC MS MS
without derivation in tobaccos
Results are comparable to our current method
The major amino acids can be analyzed within 6
minutes
Some new amino acids can be better detected
A lot of minor amino acids are Burley markers
Ala, Trp, His, Val, Lys help discriminating tobacco
types
Study the interest of the minor amino acids for the
characterization of tobaccos from the same tobacco
type (Burley)
TSRC 2003 - Norfolk