determination of tobacco specific nitrosamines (tsnas) in ...€¦ · nab nnk nnn nat nab nnk 7x104...

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

Altria Client Services l TSRC Meeting | N. Meruva l Sept. 30, 2014 l

2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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

2 Altria Client Services l TSRC Meeting | N. Meruva l Sept. 30, 2014 l

2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


TSNA Method: LC-MS/MS

NNN – 4 ng/mL

NAT – 4 ng/mL

NAB – 1 ng/mL

NNK – 4 ng/mL

2 8 4


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


Response

Higher sensitivity in positive chemical ionization (CI) mode

compared to electron ionization (EI) mode

Response


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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.


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

Analytical Workflow I


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

Analytical Workflow II


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


8 10 12


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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.


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

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


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

Questions?


2014

_TS

RC

75_S

hah.

pdf

TS

RC

2014

(68)

- D

ocum

ent n

ot p

eer-

revi

ewed

determination of tobacco specific nitrosamines (tsnas) in ...€¦ · nab nnk nnn nat nab nnk 7x104...

Documents