ANALYSIS OF N-NITROSODIMETHYLAMINE 2016_ST08_Ericsson.pdf (NDMA) IN SMOKELESS BY UHPLC-MS/MS

David , Johan Lindholm Swedish CORESTA, Berlin 2016-10-10

1 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA AGENDA

NDMA occurrence and formation 2016_ST08_Ericsson.pdf Background NDMA-analysis Presentation of UHPLC-MS/MS method • Sample preparation • Instrument parameters Method performance • Chromatograms & standard curve • Validation data • Reproducibility & Proficiency test Conclusions

2 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA

NDMA OCCURRENCE AND FORMATION

 Drinking water 2016_ST08_Ericsson.pdf • 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)

H C H C 3 3 N H NO - + 2 N N O H C 3 H C 1) Toxicological profile for NDMA, US public health service, 1989 3 2) Borgerding et al, Regulatory Toxicology and Pharmacology, Volume 64, Issue 3, December 2012, Pages 367–387

3 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA BACKGROUND- METHOD DEVELOPMENT UHPLC-MS/MS

Former in-house method, GC-TEA Aims of New in-house method, UHPLC- 2016_ST08_Ericsson.pdf method: MS/MS method: • Low sample capacity (10 samp/day) • Higher sample capacity • Less Selective detection • More Selective detection • External standard • Internal standard • Chlorinated solvents • No chlorinated solvents • LOQ (1.0 ng/g) • Lower LOQ • Labor-intensive sample preparation • Easier sample preparation (SPE and evaporation) • Robust and reliable method

The UHPLC-MS/MS method was developed in collaboration between Swedish Match and

Eurofins

4 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA SAMPLE PREPARATION

1.3-5 g of Tobacco in 100 ml E-flask 2016_ST08_Ericsson.pdf 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

5 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA UHPLC-PARAMETERS

UHPLC-Column: Waters Aquity UPLC HSS T3, 2.1mm x 150 mm,1.8µm 2016_ST08_Ericsson.pdf Mobile Phase A: MilliQ-water Mobile Phase B: Acetonitrile (ACN) Column wash: Two wash programs with Flow rate & Column temp: 0.45 ml/min & 45ºC 2-propanol (IPA), Acetonitrile and MilliQ-water Wash programs every 12th sample Injection vol: 20 µl

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

Time (min) Water % ACN% Time Water IPA Time Flow Water ACN (min) % % (min) (ml/min) % % Initial 100 0 Initial 50 50 3,0 100 0 Initial 0.1 0 100 2,0 50 50 3,6 0 100 2,0 0,1 0 100 5,0 0 100 4,6 0 100 5,0 0,1 0 100 90 0 100 5,0 100 0 10,0 0,1 0 100 105 100 0 9,0 100 0 20,0 0,2 100 0 115 100 0 25,0 0,4 100 0 130 100 0

6 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA MS/MS PARAMETERS

Mass spectrometer: Triple Quadrupole (Tandem MS) 2016_ST08_Ericsson.pdf

Ionization: APCI

Mode: Positive mode

Analyte Precursor ion Product ion (amu) (amu) NDMA 75.1 43.0 (quantifier) NDMA 75.1 58.1 (qualifier) NDMA-d6 81.1 46.0

7 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA NDMA CHROMATOGRAM- PEAK SHAPE & SELECTIVITY

 Symmetrical peak and good sensitivity despite injecting 20 µl Ethyl acetate on RP UHPLC-column 2016_ST08_Ericsson.pdf  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.

2.850 8.2e5 8.0e5

7.5e5

7.0e5 Matrix interference

6.5e5

6.0e5

5.5e5

5.0e5

4.5e5

4.0e5

Intensity, cps 3.5e5

3.0e5

2.5e5 2.0e5 NDMA 1.5e5

1.0e5

5.0e4 2.598 6.077 3.403 3.524 0.0 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 8.0 Time, min

8 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA NDMA CHROMATOGRAM- MRM-TRACES

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

2.850 8.0e5 Matrix interference 2016_ST08_Ericsson.pdf

6.0e5 Quantifier MRM = 75.1/43.0 4.0e5 NDMA

2.0e5In te n sity, cp s 2.598 3.403 3.524 6.077 0.0 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 8.0 Time, min XIC of +MRM (3 pairs): 75.1/58.1 amu from Sample 36 (st1) of 080626.wiff (Heated Nebulizer) Max. 9760.0 cps.

2.599 9760 8000 Qualifier MRM = 75.1/58.1 6000

4000

In te n sity, cp s 2000 1.097 0.009 0.424 0.952 1.316 1.781 1.898 2.158 2.730 3.407 6.125 6.396 6.673 7.181 7.634 7.830 0 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 8.0 Time, min XIC of +MRM (3 pairs): 81.1/46.0 amu from Sample 36 (st1) of 080626.wiff (Heated Nebulizer) Max. 1.2e6 cps.

2.551 1.18e6 1.00e6 8.00e5 6.00e5 NDMA-d6 (ISTD) MRM = 81.1/46.0 4.00e5

In te n sity, cp s 2.00e5 0.00 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 8.0 Time, min

9 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA STANDARD 0.1 NG/ML 2016_ST08_Ericsson.pdf Vault switch

Zoom of chromatogram above

r.t. = 2.5 min

10 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA STANDARD CURVE 2016_ST08_Ericsson.pdf

Range: 0.1-10 ng/ml Accuracy : 100-103 % R2 = 0.999

11 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA VALIDATION SUMMARY

Validation Performed with , Moist snuff, 2016_ST08_Ericsson.pdf , Raw tobacco and Nasal snuff

Repeatability: RSD 1.7% Accuracy (spiked sample): 90-104% Precision within lab: RSD 13.8% LOD: 0.2 ng/g tobacco LOQ: 0.6 ng/g tobacco

ISO 17025 Accredited

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

12 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA REPRODUCIBILITY OF METHOD

Swedish Match and Eurofins (Sweden) uses the same method 2016_ST08_Ericsson.pdf Good reproducibility between labs

Product Swedish Match Eurofins (3 replicates) (ng/g) (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

13 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA PROFICIENCY TEST

3 samples was sent from Swedish Match and analyzed as “unknowns” at 5 other labs 2016_ST08_Ericsson.pdf . 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

Product 3 replicates Swedish Match Eurofins Lab 3 Lab 4 Lab 5 Lab 6 (ng/g) UHPLC-MS/MS UHPLC-MS/MS GC-MS/MS GC-MS GC-MS ? Moist snuff A 1.3 1.4

Moist snuff B 1.0 1.0

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

14 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA ROUTINE USE OF THE METHOD

Over 10000 samples analyzed over the last 7 years 2016_ST08_Ericsson.pdf

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

15 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA CONCLUSIONS

UHPLC-MS/MS method 2016_ST08_Ericsson.pdf •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

16 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA THE END

Thanks to Johan Lindholm, David 2016_ST08_Ericsson.pdf Kälvö, Johan Patring and Birgitta Grandin at Swedish Match

Thanks to Paula Friman Eurofins, Sweden for collaboration in the method development

17 October 10, 2016 Congress2016 - Document not peer-reviewed by CORESTA