ANALYSIS OF N-NITROSODIMETHYLAMINE (NDMA) IN SMOKELESS TOBACCO BY

UHPLC-MS/MS

1

David Ericsson, Johan Lindholm

Swedish Match CORESTA, Berlin

2016-10-10

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AGENDA

NDMA occurrence and formation

Background NDMA-analysis

Presentation of UHPLC-MS/MS method

• Sample preparation

• Instrument parameters

Method performance

• Chromatograms & standard curve

• Validation data

• Reproducibility & Proficiency test

Conclusions

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NDMA OCCURRENCE AND FORMATION

Drinking water

• industrial contamination

• by-product from water treatment

Cured and/or smoked meat (up to 44 ng/g in bacon)1)

Beer (5 ng/g limit US FDA)

Tobacco and tobacco products (up to 40 ng/g)2)

GothiaTek limit 5 ng/g (~2.5 ng/g as-is)

3

+ H

3C

H3C

N H

H3C

H3C

N N O NO2

-

1) Toxicological profile for NDMA, US public health service, 1989

2) Borgerding et al, Regulatory Toxicology and Pharmacology, Volume 64, Issue 3, December 2012, Pages 367–387

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BACKGROUND- METHOD DEVELOPMENT UHPLC-MS/MS

Former in-house method, GC-TEA

method:

• Low sample capacity (10 samp/day)

• Less Selective detection

• External standard

• Chlorinated solvents

• LOQ (1.0 ng/g)

• Labor-intensive sample preparation

(SPE and evaporation)

Aims of New in-house method, UHPLC-

MS/MS method:

• Higher sample capacity

•More Selective detection

• Internal standard

• No chlorinated solvents

• Lower LOQ

• Easier sample preparation

• Robust and reliable method

4

The UHPLC-MS/MS method was developed in collaboration

between Swedish Match and

Eurofins Sweden

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SAMPLE PREPARATION

1.3-5 g of Tobacco in 100 ml E-flask

2.NDMA-d6 as ISTD (0.5 ml, 200 ng/ml)

3.5 ml MilliQ-water and shake to wet sample

4.15 ml Ethyl acetate

5.Orbital shaking for 40 min

6.Pour extract into centrifuge-tubes

7.Centrifuge 10 min (2500 rpm)

8.Transfer 1 ml of ethyl acetate to LC-vial

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UHPLC-PARAMETERS

UHPLC-Column: Waters Aquity UPLC HSS T3, 2.1mm x 150 mm,1.8µm

Mobile Phase A: MilliQ-water

Mobile Phase B: Acetonitrile (ACN)

Flow rate & Column temp: 0.45 ml/min & 45ºC

Injection vol: 20 µl

Analysis Gradient Column wash 1 (Flow 0.1 ml/min) Column wash 2

6

Time (min) Water % ACN%

Initial 100 0

3,0 100 0

3,6 0 100

4,6 0 100

5,0 100 0

9,0 100 0

Time

(min)

Water

%

IPA

% Initial 50 50

2,0 50 50

5,0 0 100

90 0 100

105 100 0

115 100 0

130 100 0

Time

(min)

Flow

(ml/min)

Water

%

ACN

%

Initial 0.1 0 100

2,0 0,1 0 100

5,0 0,1 0 100

10,0 0,1 0 100

20,0 0,2 100 0

25,0 0,4 100 0

Column wash: Two wash programs with

2-propanol (IPA), Acetonitrile and MilliQ-water

Wash programs every 12th sample

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MS/MS PARAMETERS

7

Mass spectrometer: Triple Quadrupole (Tandem MS)

Ionization: APCI

Mode: Positive mode

Analyte Precursor ion

(amu)

Product ion

(amu)

NDMA

(quantifier)

75.1 43.0

NDMA

(qualifier)

75.1 58.1

NDMA-d6 81.1 46.0

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NDMA CHROMATOGRAM- PEAK SHAPE & SELECTIVITY

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Symmetrical peak and good sensitivity despite injecting 20 µl Ethyl acetate on RP UHPLC-column

Good selectivity: Separation from interfering peak XIC of +MRM (3 pairs): 75.1/43.0 amu from Sample 36 (st1) of 080626.wiff (Heated Nebulizer) Max. 8.2e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.2e5

In

te

ns

ity

, c

ps

2.850

2.598

6.0773.403 3.524

Matrix interference

NDMA

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NDMA CHROMATOGRAM- MRM-TRACES

9

XIC of +MRM (3 pairs): 75.1/43.0 amu from Sample 36 (st1) of 080626.wiff (Heated Nebulizer) Max. 8.2e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

In

te

ns

it

y,

c

ps

2.850

2.598 6.0773.403 3.524

XIC of +MRM (3 pairs): 75.1/58.1 amu from Sample 36 (st1) of 080626.wiff (Heated Nebulizer) Max. 9760.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min

0

2000

4000

6000

8000

9760

In

te

ns

it

y,

c

ps

2.599

1.0971.316 2.730 7.8300.952 1.781 6.1251.898 2.1580.009 6.6730.424 7.181 7.6346.3963.407

XIC of +MRM (3 pairs): 81.1/46.0 amu from Sample 36 (st1) of 080626.wiff (Heated Nebulizer) Max. 1.2e6 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min

0.00

2.00e5

4.00e5

6.00e5

8.00e5

1.00e6

1.18e6

In

te

ns

it

y,

c

ps

2.551

NDMA-d6 (ISTD) MRM = 81.1/46.0

Qualifier MRM = 75.1/58.1

Quantifier MRM = 75.1/43.0

NDMA

Matrix interference

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STANDARD 0.1 NG/ML

10

r.t. = 2.5 min

Vault switch

Zoom of chromatogram above

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STANDARD CURVE

11

Range: 0.1-10 ng/ml Accuracy : 100-103 %

R2 = 0.999

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VALIDATION SUMMARY

Repeatability: RSD 1.7%

Precision within lab: RSD 13.8%

Accuracy (spiked sample): 90-104%

LOD: 0.2 ng/g tobacco

LOQ: 0.6 ng/g tobacco

12

ISO 17025

Accredited

Validation Performed with Snus, Moist snuff,

Chewing tobacco, Raw tobacco and Nasal snuff

Measurement Range: 0.1-10 ng/ml corresponding to 0.6-50 ng/g tobacco

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Swedish Match and Eurofins (Sweden) uses the same method

Good reproducibility between labs

REPRODUCIBILITY OF METHOD

13

Product

(3 replicates)

Swedish Match

(ng/g)

Eurofins

(ng/g)

Moist snuff A 1.3 1.4

Moist snuff A 1.2 1.3

Moist snuff A 1.4 1.4

Moist snuff B 1.0 1.0

Moist snuff B 0.9 1.0

Moist snuff B 1.0 1.0

Moist snuff C 20 19

Moist snuff C 17 19

Moist snuff C 19 19

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PROFICIENCY TEST

3 samples was sent from Swedish Match and analyzed as “unknowns” at 5 other labs

4 labs Z-score rating Good for Moist snuff C

Only the UHPLC-MS/MS method was sensitive enough to quantify Moist snuff A and B

14

Product

3 replicates

(ng/g)

Swedish Match

UHPLC-MS/MS

Eurofins

UHPLC-MS/MS

Lab 3

GC-MS/MS

Lab 4

GC-MS

Lab 5

GC-MS

Lab 6

?

Moist snuff A 1.3 1.4 <LOQ <LOQ <LOQ <LOQ

Moist snuff A 1.2 1.3 <LOQ <LOQ <LOQ <LOQ

Moist snuff A 1.4 1.4 <LOQ <LOQ <LOQ <LOQ

Moist snuff B 1.0 1.0 <LOQ <LOQ <LOQ <LOQ

Moist snuff B 0.9 1.0 <LOQ <LOQ <LOQ <LOQ

Moist snuff B 1.0 1.0 <LOQ <LOQ <LOQ <LOQ

Moist snuff C 19.7 19 17.8 18.7 - 13.7

Moist snuff C 17.2 19 19.4 16.4 - 16.4

Moist snuff C 18.7 19 21.7 18.4 - 15.1

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ROUTINE USE OF THE METHOD

Over 10000 samples analyzed over the last 7 years

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RSD of Control sample (Moist snuff A) = 10,3%

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CONCLUSIONS

16

UHPLC-MS/MS method

•Easier sample preparation (No SPE or evaporation)

•Higher sample capacity (30 vs. 10 samples/day)

•Excellent selectivity

•No chlorinated solvents

•Lower LOQ (0.6 ng/g vs. 1.0 ng/g)

•Good reproducibility

•Could measure in many types of smokefree tobacco

•Reliable and Robust method: Over 10000 samples

analyzed over 7 years

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THE END

Thanks to Paula Friman Eurofins,

Sweden for collaboration in the

method development

17 October 10, 2016

Thanks to Johan Lindholm, David

Kälvö, Johan Patring and Birgitta

Grandin at Swedish Match

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