Determination of ethyl carbamate in tobacco and smokeless ... · • single point external standard calibration (1000 ng/g) ... • Efficient purification method for a highly polar

JTI-Ökolab

Vienna, Austria

Herwig Stepan, Jutta Pani, Bernhard Mayer-Helm

2014 CORESTA CONGRESS - ST61

Québec City, Canada – 12-16 October 2014

Determination of ethyl carbamate in tobacco and smokeless tobacco products by HPLC-APCI+-MS/MS

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© Copyright JTI

• ethyl ester of carbamic acid

• also known as urethane

• IARC classification 2A

probably carcinogenic to humans

• listed on a draft list by the FDA for harmful and potentially

harmful constituents in tobacco products and tobacco

smoke

Determination of ethyl carbamate in tobacco and smokeless tobacco products 2

General information

Background

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Fermented beverages

• Wine

• Beer

• Spirits

• …

Fermented foods

• Soy sauce

• Vinegar

• Bread

• …

Determination of ethyl carbamate in tobacco and smokeless tobacco products

Occurrence

3

Background

Tobacco and cigarette mainstream smoke

[Schmeltz, I., Chiong, K. G., Hoffmann, D., J. Anal. Toxicol.

1978, 2, 265-268.]

Smokeless tobacco products

[Faizi A., Kimpton H., Rodu B., McAdam K., Ethyl carbamate

levels in US and Swedish smokeless tobacco products, Poster

SSPTPOST14; CORESTA Conference, Edinburgh, September 2010]

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© Copyright JTI Determination of ethyl carbamate in tobacco and smokeless tobacco products

Formation in food

4

Background

Based on [Z. Jiao et al., Compr. Rev. Food Sci. Food Saf. 2014, 13, 611-626].

diethyl pyrocarbonate

urea carbamoyl phosphate

citrulline

isocyanic acid

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Analytical methodology

5

Background

GC

ECD

EI-MS

FID NPD

TEA

PCI-MS MS/MS

HS-SPME

SPE

SLLE

Filtration

Derivatization

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Analytical methodology

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Background

LC

APCI-MS/MS FLD

ESI-MS/MS

Direct injection

SPE

Derivatization

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Analytical column: Synergi 4 µm Hydro-RP; 150 ×2 mm (Phenomenex Inc.)

Mobile phase A: 0.1% aqueous formic acid

Mobile phase B: methanol/acetonitril (50/50; v/v)

Flow rate: 0.4 ml/min

Column temperature: 30 °C

Injection volume: 20 µl


LC parameters

7

LC-MS/MS analysis

Time (min) Mobile phase A (%) Mobile phase B (%)

0.0 100 0

6.0 100 0

6.5 0 100

9.5 0 100

10.0 100 0

15.0 100 0

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Infusion of standard solution

Precursor Ion m/z 90.0

Transitions 90.062.0

90.044.0

90.029.0

62.029.0


MS parameters

8

LC-MS/MS analysis

C2H4

+H

+

+H

+

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Infusion of standard solution

Precursor Ion m/z 90.0

Transitions 90.062.0

90.090.0


MS parameters

9

LC-MS/MS analysis

C2H4

+H

+

+H

+

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Precursor

ion

(m/z)

Product ion

(m/z)

Declustering

potential

(V)

Entrance

potential (V)

Collision

energy

(V)

Cell exit

potential (V)

Quantifier 90.0 62.0 36 10 12 7

Qualifier 90.0 90.0 36 10 5 8

EC-d5 95.0 63.0 36 10 12 7


MS parameters

10

LC-MS/MS analysis

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ESI vs. APCI

11

Ionization technique

ESI APCI Enhancement

Peak area 90.062.0 5.9 × 106 2.1 × 107 3.6 ×

90.090.0 5.5 × 106 1.6 × 107 2.8 ×

S/N ratio 90.062.0 105 482 4.6 ×

90.090.0 43 203 4.7 ×

Instrument-LOQ (ng/mL) 23.2 4.9 4.7 ×

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• 1 g tobacco or smokeless tobacco product

Sample

• Addition of internal standard (EC-d5)

Spiking • 10 mL 0.1% aqueous formic

acid

• 10 min ultrasonic bath

• 20 min shaker

Extraction


Sample extraction

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0

200000

400000

600000

800000

3 4 5 6 7

Inte

nsit

y (

cp

s)

Time (min)

m/z 90.062.0

Recommendation according to EC SANCO 12571/2013:

Identification of analyte using MS/MS: at least 2 transitions

Calculation of LOQ / LOD using S/N from the second best transition


Direct injection

13

LC-MS/MS analysis

LOQ ~ 100 ng/g

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0

200000

400000

600000

800000

3 4 5 6 7

Inte

nsit

y (

cp

s)

Time (min)

m/z 90.062.0

Recommendation according to EC SANCO 12571/2013:

Identification of analyte using MS/MS: at least 2 transitions

Calculation of LOQ / LOD using S/N from the second best transition

0

200000

400000

600000

800000

3 4 5 6 7

Inte

nsit

y (

cp

s)

Time (min)

m/z 90.090.0

m/z 90.062.0


Direct injection

14

LC-MS/MS analysis

LOQ > 1500 ng/g

LOQ ~ 100 ng/g

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Calculation of partition ratio

log P (ethyl carbamate) = -0.15

• log P > 1: binding to silica-based C18

• log P < -1: usually no binding to RP material

separation of analytes with log P between -1 and 1?


Reversed-phase solid phase extraction (RP-SPE)

15

Selective extraction of EC

log 𝑃 𝑜𝑐𝑡𝑎𝑛𝑜𝑙

𝑤𝑎𝑡𝑒𝑟= log

𝐴𝑛𝑎𝑙𝑦𝑡𝑒 𝑜𝑐𝑡𝑎𝑛𝑜𝑙

𝐴𝑛𝑎𝑙𝑦𝑡𝑒 (𝑢𝑛𝑖𝑜𝑛𝑖𝑧𝑒𝑑) 𝑤𝑎𝑡𝑒𝑟

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Polymeric RP-SPE

16


Polymeric sorbents (e. g. N-vinylpyrrolidone)

multiple modes of retention for stronger analyte-sorbent interaction

allow binding of analytes with partition coefficients as low as -1

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Comparison of conventional C18 and polymeric RP sorbents

17


0

20

40

60

80

100

120

Standard Matrix Standard Matrix

Recovery

(%

)

Flow-through Wash Eluate 1 Eluate 2

C18 Strata-X

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• single point external standard calibration (1000 ng/g)

• correction with the internal standard EC-d5


Quantification

18

Application on smokeless tobacco reference products

𝐸𝐶 (𝑛𝑔

𝑔) =

1000 × 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝐸𝐶 𝑠𝑎𝑚𝑝𝑙𝑒 × 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝐸𝐶 − 𝑑5 (𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑)

𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝐸𝐶 − 𝑑5 𝑠𝑎𝑚𝑝𝑙𝑒 × 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝐸𝐶 (𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑)

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Determination of LOQ and LOD in reference materials:

CRP2 (moist snuff – moisture content: 55%)

CRP3 (dry snuff – moisture content: 8%)


Determination of LOQ and LOD

19


wet weight basis dry weight basis

CRP2 CRP3 CRP2 CRP3

LOQ (ng/g) 89 193 196 210

LOD (ng/g) 27 58 59 63

Method-LOQ/LOD

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Smokeless tobacco reference products + reference cigarette filler

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Description Value

CRP1 Swedish-style snus pouch < LOD

CRP2 American-style loose moist snuff

CRP3 American-style loose dry snuff powder < LOD

CRP4 American-style loose-leaf chewing tobacco < LOD

3R4F filler Kentucky Reference Cigarette < LOD


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Smokeless tobacco reference products + reference cigarette filler

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Description Value

CRP1 Swedish-style snus pouch < LOD

CRP2 American-style loose moist snuff < LOQ 37.5 ng/g (WWB)

CRP3 American-style loose dry snuff powder < LOD

CRP4 American-style loose-leaf chewing tobacco < LOD



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MRM chromatogram of CRP2

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0

50000

100000

150000

0 1 2 3 4 5 6 7

Inte

nsit

y (

cp

s)

Time (min)

EC

m/z 90.062.0

m/z 90.090.0

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30 smokeless tobacco samples (moisture content: 27-52%)

• 17 samples (57%): <LOD

• 12 samples (40%): <LOQ

• 1 sample (3%): 162 ng/g (wet weight basis)

Comparison to literature (Faizi et al., 2010):

• 40 samples (57%): <LOD (20 ng/g WWB)

• 30 samples (43%): >LOD


Snus samples

23

Results

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• Efficient purification method for a highly polar compound

significant improvement of LOQ compared to direct injection

• ESI vs. APCI

APCI is 5 times more sensitive than ESI

• Comparison with literature

Schmeltz et al. did not analyze smokeless tobacco products

accordance with recent data (Faizi et al.)


Conclusion

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© Copyright JTI 25

I want to thank my team! Herwig Stepan

Bernhard Mayer-Helm

Stefan Pummer


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Thank you for your attention!


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Determination of ethyl carbamate in tobacco and smokeless ... · • single point external standard calibration (1000 ng/g) ... • Efficient purification method for a highly polar

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