determination of tobacco specific nitrosamines (tsnas) in ...€¦ · nab nnk nnn nat nab nnk 7x104...
TRANSCRIPT
Niti Shah, Tyler Smith, Jason Flora and Naren Meruva
Determination of Tobacco Specific Nitrosamines (TSNAs) in Tobacco and Tobacco Smoke
by GC-MS/MS
1
Altria Client Services,
601 East Jackson St, Richmond, VA 23219
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Background
NNN, NNK, NAT and NAB, commonly referred as tobacco specific
nitrosamines (TSNAs) are listed as harmful and potentially harmful
constituents (HPHCs) in tobacco and tobacco smoke.
NNN → N'-nitrosonornicotine, NNK → (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone,
NAB → N'-nitrosoanabasine and NAT → N-nitrosoanatabine
NNK NNN NAT NAB
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Background – TSNA Methods
Year CORESTA Standard
Instrument Method Scope
2005 N° 63 GC-TEA Mainstream Smoke
2011 N° 72 HPLC-MS/MS Smokeless Tobacco
2012 N° 75 HPLC-MS/MS Mainstream Smoke
High sensitivity, selectivity and analysis throughput have led TSNA method standardization process
GC – Gas Chromatography; TEA – Thermal Energy Analyzer; HPLC – High
Performance Liquid Chromatography; MS – Mass Spectrometry
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TSNA Method: GC-TEA
High selectivity
Low instrumentation
cost (~$100K)
Low sensitivity
Requires sample
cleanup/concentration
NNN
NAT
NAB
NNK
50 ng/mL calibration standard
Retention Time (min)
Re
spo
nse
5 11 8
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Improved selectivity
High sensitivity
Simplified sample
preparation
High cost of
instrumentation (~$350K)
NNN – 48ng/mL
NAT – 48ng/mL
NAB – 12ng/mL
NNK – 48ng/mL
Response
Retention Time (min)
TSNA Method: LC-MS/MS
NNN – 4 ng/mL
NAT – 4 ng/mL
NAB – 1 ng/mL
NNK – 4 ng/mL
2 8 4
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Objective
Develop a more sensitive, selective and high throughput
method for determination of TSNAs in tobacco and tobacco
smoke by GC-MS/MS
Advantages:
• High sensitivity and selectivity
• Lower instrument cost (~$200k)
• Common consumables
Method Optimization:
• Injection parameters
• Ionization mode
• Sample preparation
• TSNAs yield: GC-MS/MS vs. LC-MS/MS
Agilent 7890 GC/7000 MS/MS
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Injection Mode Optimization
Solvent Vent, 5 µL Injection
S/N = 27,868
Cold Splitless, 1 µL Injection
S/N = 2744
Hot Splitless, 1 µL Injection
S/N = 456
10-fold higher sensitivity
in solvent vent mode
compared to cold
splitless mode
NNN
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Ionization Mode Optimization: EI vs. CI M + e- → M1 + M2 + M3… EI M + NH4
+ → M + 1 CI NNN
NAT
NAB
NNK NNN
NAT
NAB
NNK
7x104 1.25x105
Retention Time (min)
Response
Higher sensitivity in positive chemical ionization (CI) mode
compared to electron ionization (EI) mode
Response
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Limitations of EI Mode
• Comparable yields for NNN,
NNK and NAT using EI and
CI modes
• Poor sensitivity for NAB
detection in EI mode
• CI mode provides high
sensitivity for measurement
of all four TSNAs
Below LOQ
for NAB
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
NNN NAT NAB NNK
Co
nc. (µ
g/g
)
Electron Impact (EI)
Chemical Ionization (CI)
3R4F Filler Extract
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CI Reagent Gas Optimization
0
5000
10000
15000
20000
NAB
Peak
Are
a
Methane
5% Ammonia in Methane
Ammonia
Proton affinity of ammonia
is closest to the analytes,
resulting in minimal
fragmentation and higher
sensitivity for TSNAs.
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GC-MS/MS Analytical Method
Gas Chromatography (GC)
GC Column DB-35MS-UI (30 m x 0.25 mm ID x 0.25 µm)
Inlet Temperature
Program -10 °C for 2 min, ramp 600 °C/min to 325 °C
Injection Mode Solvent Vent
Injection Volume 5 µL
Column Flow 1 mL/min
Oven Temperature
Program
Initial 70 C hold for 2.0 min
Ramp 30 C/min to 170 C
Ramp 20 ºC/min to 250 ºC
Ramp 60 ºC/min to 300 ºC, hold for 3.0 min
GC Run Time 13.2 min
Sample preparation following Analytical Workflow II
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GC-MS/MS Analytical Method
Tandem Mass Spectrometry (CI)
Analyte/ISTD* Precursor Ion Product Ion CE* (V)
NNN 178 148 8
NAT 190 160 8
NAB 192 162 8
NNK 208 122 12
NNN-D4 182 152 8
NAT-D4 194 164 5
NNK-D4 212 126 12
Same MS/MS Transitions as LC-MS/MS method
* CE = Collision Energy * ISTD = Internal Standard
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Analytical Workflow I
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Analytical Workflow II
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TSNA Method: GC-MS/MS (CI)
NNN – 0.8 ng/mL
NNK – 0.8 ng/mL
NAB – 0.2 ng/mL
NAT – 0.8 ng/mL
High selectivity
High sensitivity
Lower cost per analysis
Simplified sample preparation
Response
Retention Time (min)
8 10 12
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TSNAs Data - Cigarette Smoke (3R4F/ISO)
TSNA yields are equivalent between
GC-MS/MS and LC-MS/MS
0
20
40
60
80
100
120
140
NNN NAT NAB NNK
Co
nc.
(ng
/cig
)
GC-MS/MS LC-MS/MS N = 6 reps; Error bars ±1 S.D.
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TSNA Data – Reference Tobacco Products
NNK NNN
TSNA yields are equivalent between
GC-MS/MS and LC-MS/MS
N = 6 reps
Error bars ±1 S.D.
N = 6 reps
Error bars ±1 S.D.
0
2
4
6
8
10
CRP-1 CRP-2 CRP-3 CRP-4 3R4F Filler
Co
nc.
(µg
/g,
as
-is
)
GC-MS/MS
LC-MS/MS
0
1
2
3
4
5
CRP-1 CRP-2 CRP-3 CRP-4 3R4F Filler
GC-MS/MS
LC-MS/MS
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Summary
GC-MS/MS method provides higher sensitivity and
selectivity for determination of TSNAs
Suitable for determination of TSNAs in snus, moist snuff,
dry snuff, chewing tobacco, tobacco filler and cigarette
smoke
TSNA yields from GC-MS/MS and LC-MS/MS are
equivalent
GC-MS/MS method reduces the cost of routine TSNA
analysis without compromising data quality
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Questions?
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