Technical Report 2020 Nicotine and Nicotine Degradants ...

Tobacco and Tobacco Products Analytes Sub-Group

Technical Report

2020 Nicotine and Nicotine

Degradants Proficiency Study

September 2020

Sub-Group Coordinator and Study Coordinator:

Karl Wagner, Ph.D., Altria Client Services LLC, U.S.A.

Author and Study Coordinator:

Fadi Aldeek, Ph.D., Altria Client Services LLC, U.S.A.

Co-Author and Statistical Analysis:

Michael Morton, Ph.D., Altria Client Services LLC, U.S.A.

Table of Contents

1. Summary ........................................................................................................................... 3

2. Introduction ....................................................................................................................... 3

3. Organization ...................................................................................................................... 3

3.1 Participants .............................................................................................................. 3

3.2 Protocol ................................................................................................................... 4

4. Data ................................................................................................................................... 5

5. Statistical Analysis ............................................................................................................ 5

6. Results – Z-Scores ............................................................................................................ 8

7. Discussion ....................................................................................................................... 14

8. Conclusions ..................................................................................................................... 15

9. Recommendations ........................................................................................................... 15

APPENDIX A: Study Protocol ............................................................................................... 16

APPENDIX B: Data Tables ................................................................................................... 20

APPENDIX C: Raw Data Plots .............................................................................................. 44

APPENDIX D: Graphs of z-scores......................................................................................... 52

APPENDIX E: Summary of Method Synopses for Nicotine Analysis .................................. 59

APPENDIX F: Summary of Method Synopses for Nicotine Degradants Analysis

in NP Samples .............................................................................................. 60

APPENDIX G: Summary of Method Synopses for Nicotine Degradants Analysis

in EL Samples .............................................................................................. 61

APPENDIX H: Summary of Internal Standards used for each Analyte ................................ 62

TTPA-246-1-CTR 2020 Nicotine and Nicotine Degradants Proficiency Study – September 2020 3/62

1. Summary

The CORESTA Tobacco and Tobacco Products Analytes (TTPA) Sub-Group conducted a

proficiency study in the first quarter of 2020 for the determination of nicotine and nicotine

degradants in nicotine pouches and e-liquids. This study included five nicotine pouch and four

flavoured e-liquid samples (Appendix A). Nineteen laboratories participated in the nicotine

analysis and fifteen laboratories participated in the nicotine degradants analysis.

2. Introduction

The objective of this proficiency study was to provide a continuing assessment of laboratory

capabilities for the analysis of nicotine and nicotine degradants in nicotine pouches and

e-liquids. Nicotine pouches are tobacco leaf-free products that contain tobacco derived nicotine.

In order to limit the influence of sample homogeneity on the results, bulk nicotine pouch filler

was distributed to the participating laboratories. Market-like, flavoured e-liquid samples were

fortified as stated below in the protocol section before distribution to the participating

laboratories. Laboratories were requested to determine nicotine, anatabine, anabasine, nicotine-

N’-oxide, myosmine, ß-nicotyrine, cotinine, and nornicotine using their in-house methodologies.

3. Organization

3.1 Participants

A list of the participating laboratories is provided in Table 1. The laboratory codes used in this

report do not correspond to the same order as provided in Table 1.

Table 1: List of Participating Laboratories

Participating Laboratories

Altria Client Services LLC, United States

University of Kentucky, United States

China National Tobacco Quality Supervision and Test Center, Zhengzhou, China

Enthalpy Analytical, Richmond, United States

Essentra Scientific Services, United Kingdom

Global Laboratory Services, United States

Imperial Brands, Reemtsma, Hamburg, Germany

National Institute of Standards and Technology, Gaithersburg MD, United States

Japan Tobacco Inc., Leaf Tobacco Research Center, Oyama, Japan

JTI Ökolab, Vienna, Austria

KT&G, Daejeon, South Korea

Labstat International ULC, Kitchener, Canada

Philip Morris International, Switzerland

RJ Reynolds Tobacco Company, United States

Swedish Match, Stockholm, Sweden

INCIENSA, Costa Rica

ITC Limited - Agri Business Division, India

Eurofins Food & Feed Testing Sweden AB, Lidköping, Sweden


3.2 Protocol

Specific details from the protocol are described below and the full protocol is provided in

Appendix A. This study included five market-like nicotine pouch filler samples (NP-1 to

NP-5). As mentioned earlier, filler samples were chosen instead of intact nicotine pouches so

that sample homogeneity could be ensured prior to shipment to the participants. Three of the

five filler samples (NP-2, NP-4, NP-5) were subjected to accelerated aging at elevated

temperature and humidity in an effort to generate measurable levels of analytes. Nicotine pouch

filler samples NP-1 and NP-2 were provided by Swedish Match and NP-3 through NP-5 were

provided by American Snuff Co. The samples are identified in Table 2.

Table 2: NP Samples Description

Sample ID

Description Supplier

NP-1 Nicotine pouch, homogeneous bulk sample without pouch paper Swedish Match

NP-2 Artificially aged nicotine pouch, homogeneous bulk sample without

pouch paper Swedish Match

NP-3 Nicotine pouch, homogeneous bulk sample without pouch paper American Snuff


pouch paper American Snuff


pouch paper American Snuff

The study also included four e-liquid samples (EL-1 to EL-4). The samples are identified in

Table 3. The e-liquids contained carrier (propylene glycol and glycerine), nicotine and flavours.

EL-1, EL-2, and EL-3 samples are market-like, flavoured, and nicotine free e-liquid. These

three samples were fortified with artificially aged nicotine in an effort to have measurable levels

of degradants. Additionally, EL-1 and EL-3 were fortified with nicotine degradants as described

in Table 3. Since EL-1 and EL-3 contained artificially aged nicotine and fortified nicotine

degradants, the concentration of the analytes is greater than the fortified amounts shown in

Table 3. The nicotine used in these samples was artificially aged by placing the neat nicotine in

an oven set to 40 °C for approximately 24 hours. The nicotine was then allowed to sit at room

temperature overnight before the e-liquids were made.

Sample EL-4 was a market-like e-liquid that was naturally aged for an extended period of time.

Laboratories were requested to store the samples in the freezer upon receipt to limit further

degradation. E-liquid samples (EL-1 to EL-4) were provided by Enthalpy.

Table 3: EL Samples Description

Sample ID

Propylene Glycol/Glycerin

Ratio

Approximate Nicotine Conc.

(mg/mL) Flavour

Analytes – Fortification Level

(µg/mL)1

EL-1 50/50 20 Unflavored

Anatabine – 92 Nicotine-N’-oxide – 115

Nornicotine – 142 Anabasine – 74 Myosmine – 65 Cotinine – 101

EL-2 50/50 20 Tobacco Not Fortified


Sample ID

Propylene Glycol/Glycerin

Ratio

Approximate Nicotine Conc.

(mg/mL) Flavour

Analytes – Fortification Level

(µg/mL)1

EL-3 50/50 20 Sweet

Anatabine – 30 Nicotine-N’-oxide – 38

Nornicotine – 74 Anabasine – 25 Myosmine – 22 Cotinine – 34

EL-4 50/50 40 Mint Not fortified

1. EL-1 and EL-3 contained artificially aged nicotine and fortified nicotine degradants. For this reason, the concentration

of the analytes is greater than the fortified amounts shown here.

Laboratories were requested to store the samples in the freezer upon receipt. 24 hours prior to

the analysis, laboratories were recommended to remove the samples from the freezer and place

them in the refrigerator. Laboratories were requested to remove the samples from the

refrigerator for a minimum of 2 hours prior to opening for analysis. Three independent replicate

analyses were required for each sample. The replicates were determined from independently

prepared sample aliquots. Data were to be reported in units of mg/g for nicotine and µg/g for

nicotine degradants. Laboratories were requested to submit data by March 15th; however, data

were accepted until April 30th.

4. Data

The raw data are provided in Appendix B on an as-is basis without any correction for moisture.

Graphs showing data per mg/g for nicotine and µg/g for nicotine degradants are provided in

Appendix C. Statistical analysis data are provided in Table 4. Z-score for individual and overall

data and graphs are provided in Tables 5 to 14 and Appendix D, respectively. Summary of the

methods synopses are provided in Appendices E, F, and G. Internal standard information is

provided in Appendix H.

5. Statistical Analysis

The analysis was carried out in basic conformance with ISO 13528. The assigned values and

standard deviation for proficiency assessment for each compound, and for each sample, were

developed using the robust Algorithm A. The standard deviations were also compared to values

using the Horwitz equation as a benchmark to determine if the calculated standard deviations

were within the expected range. The assigned values and standard deviations were only

calculated if there were at least eight laboratories with quantified results for a sample. It

appeared that there was a sample mix-up in the EL samples in Lab 11, so Lab 11 was omitted

in the calculation of the EL assigned values, standard deviations, and count of labs with

quantified values; however, Lab 11 was included in the z-statistic calculations.


Table 4: Data Summary (as-is basis)

NP-1

Nicotine (mg/g)

Anabasine (µg/g)

Anatabine (µg/g)

β-Nicotyrine (µg/g)

Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

16,1 NA NA NA NA NA NA NA

Standard deviation


Standard deviation Horwitz


NP-2

Nicotine (mg/g)

Anabasine (µg/g)

Anatabine (µg/g)


Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

13,1 NA NA NA NA NA NA 5,49

Standard deviation




NP-3

Nicotine (mg/g)

Anabasine (µg/g)

Anatabine (µg/g)


Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

8,94 NA NA NA 2,22 2,39 46,6 4,59

Standard deviation

0,72 NA NA NA 0,45 1,0 23,2 4,37


0,36 NA NA NA 0,32 0,34 4,2 0,58

NP-4

Nicotine (mg/g)

Anabasine (µg/g)

Anatabine (µg/g)


Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

8,68 NA NA NA 3,65 2,75 502 7,14

Standard deviation

1,05 NA NA NA 0,48 1,14 129 5,67


0,35 NA NA NA 0,48 0,38 31,5 0,85

NP-5

Nicotine (mg/g)

Anabasine (µg/g)

Anatabine (µg/g)


Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

8,75 NA NA NA 8,47 4,13 1116 10,2

Standard deviation

0,91 NA NA NA 1,12 1,44 296 9,2


0,36 NA NA NA 0,98 0,53 62,1 1,15


EL-1

Nicotine

(mg/g)

Anabasine (µg/g)

Anatabine

(µg/g)


Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

12,6 41,7 87,1 NA 44,9 44,5 66,5 37,4

Standard deviation

1,19 7,94 23,4 NA 11,4 6,5 24,7 16,1


0,49 3,81 7,12 NA 4,05 4,02 5,7 3,47

EL-2

Nicotine

(mg/g)

Anabasine

(µg/g) Anatabine

(µg/g) β-Nicotyrine

(µg/g)

Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

12,9 18,2 60,0 NA 18,7 26,3 23,2 7,98

Standard deviation

1,41 2,45 17,42 NA 1,73 2,44 11,8 5,9


0,50 1,88 5,19 NA 1,93 2,57 2,3 0,93

EL-3

Nicotine

(mg/g)

Anabasine

(µg/g) Anatabine

(µg/g)

β-Nicotyrine

(µg/g)

Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

13,0 85,7 137 NA 93,9 79,3 139 119

Standard deviation

1,37 9,00 39,85 NA 29,9 9,4 23,2 36,8


0,50 7,02 10,46 NA 7,58 6,57 10,6 9,28

EL-4

Nicotine

(mg/g)

Anabasine

(µg/g)

Anatabine

(µg/g)

β-Nicotyrine

(µg/g)

Cotinine (µg/g)

Myosmine (µg/g)

Nicotine-N’-Oxide

(µg/g)

Nornicotine (µg/g)

Assigned Value

35,0 NA NA NA 15,4 19,9 214 24,9

Standard deviation

3,85 NA NA NA 1,8 4,6 44,2 24,1


1,16 NA NA NA 1,63 2,03 15,2 2,45

The assigned values and standard deviations were estimated using Algorithm A. For comparison, the standard deviations

were also calculated from the Horwitz equation.

“NA” signifies the z-score was not calculated because fewer than eight laboratories with quantified values were provided.

The calculated standard deviations tend to be noticeably larger than those predicted by the

Horwitz equation. The means and standard deviations are calculated using a robust statistical

algorithm which is resistant to around 25 % outliers. The observed larger than expected standard

deviations could be attributed to the different analytical and extraction methods used by the

participating labs. Method standardization could improve the agreement among laboratories.


6. Results – Z-Scores

The corresponding z-statistics are given in Tables 5–13. The z-scores are calculated as

z = (x-μ)/σ, where μ and σ are the estimated means and standard deviations, respectively. As

shown in Table 4, the x is the average value from the labs for each analyte and sample.

Generally, the z-scores are regarded as reasonable if -2 < z < 2 and thought to be unacceptable

if |z| > 3. Laboratories should consider |z| between 2 and 3 as a warning signal. Laboratories

should particularly be aware of the pattern of their results. For example, a single z-value signal

should be investigated, but a pattern of problematic z-scores in the same direction for the same

analyte should be taken much more seriously. Graphs of the z-scores are shown in Appendix D.

Under ideal circumstances in which the mean and standard deviation are known quantities,

around 95 % of the z-scores will have |z| < 2, around 5 % will have 2 < |z| < 3 and around

0,27 % will have |z| >3. After calculation of the mean and standard deviation, one would expect

more than 95 % of the results to have |z| < 2 because the mean and standard deviation were

calculated to fit the data. A separate simulation study (data not shown) was carried out to

investigate the z-score calculations being based on the experimental means and standard

deviations. The simulation showed that if the data of n=10 labs were used, around 97 % of the

labs should have |z| < 2, 2,5 % should have 2 < |z| <3, and 0,53 % should have |z| >3.

Table 5: Z-Score Results for NP-1

Lab Code

Nicotine Anabasine Anatabine β-Nicotyrine Cotinine Myosmine Nicotine-N’-Oxide

Nornicotine

1 0,18 NA NA NA NA NA NA NA

2 -2,15 NA NA NA NA NA NA NA
















18 NA NA NA NA NA NA NA NA





Lab Code


Nornicotine




4 0,74 NA NA NA NA NA NA -0,82

5 -1,80 NA NA NA NA NA NA 2,56


7 0,05 NA NA NA NA NA NA 1,10


9 -0,73 NA NA NA NA NA NA -0,78


11 0,97 NA NA NA NA NA NA -0,82








19 2,19 NA NA NA NA NA NA -0,10



Lab Code

Nicotine Anabasine Anatabine β-Nicotyrine Cotinine Myosmine Nicotine-N’-

Oxide Nornicotine

1 -0,31 NA NA NA -0,71 -0,49 -0,79 NA



4 -0,10 NA NA NA 0,13 -0,71 -0,61 -0,85

5 0,30 NA NA NA NA 1,41 NA 2,69

6 0,53 NA NA NA 1,00 1,20 -0,23 NA

7 -0,31 NA NA NA 0,95 -0,56 0,93 1,22


9 -1,42 NA NA NA 0,61 -0,28 0,93 -0,72

10 0,01 NA NA NA NA NA -0,85 NA

11 -0,45 NA NA NA 0,22 -0,56 1,81 -0,73

12 0,48 NA NA NA -1,72 -0,58 NA -0,77




Lab Code

Nicotine Anabasine Anatabine β-Nicotyrine Cotinine Myosmine Nicotine-N’-

Oxide Nornicotine


16 0,83 NA NA NA NA 1,19 NA NA

17 7,57 NA NA NA 0,31 -0,62 -0,66 0,27


19 2,86 NA NA NA -1,02 NA -0,20 -0,10



Lab Code


Nornicotine

1 -0,32 NA NA NA -0,92 -0,57 -0,92 -0,74

2 -0,20 NA NA NA NA NA NA 7,12


4 -0,15 NA NA NA 0,38 -0,69 -1,02 -0,79

5 0,06 NA NA NA NA 1,11 NA 1,80

6 0,18 NA NA NA 0,98 1,17 0,28 NA

7 -0,63 NA NA NA 0,66 -0,58 2,35 0,59


9 -1,09 NA NA NA 0,71 -0,31 0,11 -0,62

10 -0,11 NA NA NA NA NA -0,59 NA

11 -0,24 NA NA NA 0,52 -0,53 -0,71 -0,78

12 1,08 NA NA NA -2,85 -0,40 NA -0,73





17 3,78 NA NA NA 0,07 -0,69 1,02 0,25


19 1,54 NA NA NA -0,90 NA 0,33 -0,38



Lab Code


Nornicotine

1 -0,30 NA NA NA -0,86 -0,59 -0,89 -0,63

2 -0,13 NA NA NA NA NA NA 13,96


4 -0,19 NA NA NA 0,12 -0,81 -1,00 -0,71

5 0,16 NA NA NA 1,48 0,72 NA 1,44

6 0,65 NA NA NA 0,57 1,12 0,41 NA


Lab Code


Nornicotine

7 -0,89 NA NA NA 0,06 -0,76 1,65 0,20


9 -0,72 NA NA NA 0,63 -0,30 0,11 -0,54

10 -0,18 NA NA NA NA NA -0,22 NA

11 0,38 NA NA NA 0,04 -0,71 -0,72 -0,78

12 1,28 NA NA NA -2,78 -0,17 NA -0,60







19 2,21 NA NA NA -0,55 NA 0,81 -0,43


Table 10: Z-Score Results for EL-1

Lab Code


Nornicotine

1 0,17 0,08 -0,88 NA -0,14 0,67 -0,73 -0,49



4 -0,03 -0,44 -0,05 NA 0,27 -0,29 -0,77 -0,21

5 0,88 1,33 2,53 NA 0,79 0,18 NA 3,32

6 0,22 0,54 0,44 NA 0,53 1,05 0,04 3,63

7 -1,04 -0,10 0,04 NA 0,52 -0,07 2,00 0,53

8 0,54 -0,91 -3,42 NA -3,73 -5,72 NA 0,56

9 -0,88 -0,74 0,68 NA 0,01 0,36 -0,39 -0,34

10 0,26 0,27 -0,28 NA 0,13 -0,01 -0,57 -0,73

11 21,91 NA NA NA -2,69 -4,10 4,33 -1,77





16 -0,48 0,49 0,23 NA -2,23 2,04 NA -0,68

17 1,13 1,45 1,19 NA -0,72 -1,27 1,23 0,12

18 1,09 -1,52 -0,94 NA 1,63 -0,79 NA -1,38

19 3,27 -0,47 -0,44 NA 0,11 0,17 -0,31 -0,37




Lab Code Nicotine Anabasine Anatabine β-Nicotyrine Cotinine Myosmine Nicotine-N’-Oxide

Nornicotine

1 0,23 0,31 -0,96 NA -0,95 0,30 -0,74 -0,63



4 0,15 -0,28 -0,16 NA 0,72 -0,28 -0,93 -0,84

5 0,64 3,50 2,43 NA NA 0,31 NA 6,29

6 0,13 3,50 0,42 NA 1,15 1,28 -0,01 NA

7 -0,88 -0,50 -0,45 NA -0,11 -1,09 1,19 1,22

8 0,18 -1,08 NA NA NA NA NA 0,20

9 -1,13 -0,83 0,31 NA -0,25 -0,01 -0,36 -0,88

10 0,29 0,30 -0,40 NA -0,15 -0,38 -0,17 NA

11 -6,54 9,33 1,32 NA 15,80 7,92 2,89 2,14





16 -0,43 0,78 -0,01 NA NA 2,61 NA NA

17 3,93 -0,32 5,52 NA 1,07 0,05 3,68 0,29

18 0,87 -0,69 -1,16 NA -1,87 -1,63 NA -0,69

19 2,62 -0,70 -0,59 NA 0,03 -0,18 -0,47 -0,17



Lab Code


Nornicotine

1 0,27 0,22 -0,55 NA -0,12 0,60 -0,80 -0,58



4 -0,10 -0,59 0,15 NA 0,42 0,00 -0,72 -0,24

5 0,65 1,18 2,36 NA 0,79 -0,06 NA 1,83

6 0,13 0,18 0,60 NA 0,37 1,17 0,78 1,25

7 -0,76 -0,13 0,23 NA 0,46 -0,67 4,31 -0,28

8 0,11 -0,52 -2,20 NA -1,65 -4,37 NA 1,12

9 -0,41 -1,20 0,86 NA 0,15 0,29 -0,30 -0,27

10 0,13 0,48 0,12 NA 0,25 0,05 0,13 -0,64

11 -6,36 -7,50 -2,16 NA -2,53 -5,71 -5,10 NA






Lab Code


Nornicotine

16 -0,40 1,03 0,47 NA -1,71 2,40 NA -0,76

17 9,58 1,18 -1,77 NA -0,95 -1,12 -1,05 0,55

18 1,09 -2,24 -0,30 NA 1,36 -0,63 NA -1,20

19 2,90 -0,30 -0,08 NA 0,28 0,36 0,46 -0,46



Lab Code


Nornicotine

1 0,45 NA NA NA -0,67 -0,53 -0,87 -0,73



4 0,47 NA NA NA 0,56 -0,55 -0,83 -0,72

5 0,91 NA NA NA NA 1,50 NA 1,97

6 0,31 NA NA NA 1,20 0,40 0,22 NA

7 0,30 NA NA NA -0,05 -0,90 3,68 0,80


9 -0,65 NA NA NA -0,51 -0,43 -0,37 -0,67

10 0,68 NA NA NA -0,58 -0,57 -0,86 NA

11 -7,70 NA NA NA 50,66 14,54 -1,36 2,45





16 -2,57 NA NA NA NA 0,73 NA NA

17 -4,22 NA NA NA 9,52 2,84 0,80 1,12

18 0,63 NA NA NA -0,97 -0,43 NA -0,42

19 2,56 NA NA NA -0,48 -0,71 0,42 -0,55


The overall results of the proficiency study, showing the percentage of acceptable results where

|z| ≤ 2, are given in Table 14.


Table 14: Overall Proficiency Study Results

Analyte Number of Scores

|z| ≤ 2 Total Number of

Scoresǂ Percent |z| ≤ 2 ǂ

Nicotine 131 156 84,0

Anabasine 33 38 86,8

Anatabine 30 37 81,1

β-Nicotyrine NA NA NA

Cotinine 64 73 87,7

Myosmine 69 79 87,3

Nicotine-N’-Oxide 54 62 87,1

Nornicotine 74 83 89,2

ǂ: Laboratories that reported <LOD, <LOQ or did not determine the element in the matrix were not included in the sum

nor the percentage.


As discussed above, less than 5 % of the labs are expected to have an absolute z-score greater

than 2; however, these results exceeded that percentage. Furthermore, multiple large z-scores

(i.e. |z| > 4) values were calculated (Tables 5-13) for many analytes. It is expected that a method

standardization would improve these results.

7. Discussion

All 19 labs participated in the nicotine analysis whereas 15 labs provided data for nicotine

degradants. From the 15 participating labs in the nicotine degradants analysis, 5 labs did not

analyse for nicotine-N’-oxide while three labs did not analyse β-nicotyrine. In addition, Lab 2

did not analyse for cotinine in samples (Appendix D).

Despite the different extraction and analytical methods used by the 19 participant labs for the

determination of nicotine, the nicotine results provided by most of the participating labs were

relatively consistent except for lab 11 and 17 in some samples (Appendix B and C). We believe

that EL samples may have been switched for lab 11.

Anabasine and anatabine were not detected in the NP samples by any labs. These two analytes

were detected in EL samples by all labs except for lab 2. However, anabasine and anatabine in

EL-4 were found to be below the LOQ of some participating labs. Anabasine and anatabine in

EL-4 were determined by a few labs that used a combination of low method LOQ and

isotopically labelled internal standards for these analytes.

Mysomine, nornicotine, and cotinine in EL samples were detected by most participating labs

except lab 2. Cotinine and mysomine in NP samples were below the method LOQ of some labs

(e.g., lab 8 and 10). Similarly, nornicotine was found to be below the method LOQ of some labs

(e.g., lab 6, 10, and 16).

Nicotine-N’-oxide was not determined in all samples by Lab 2, 5, 8, 12, and 16. This could be

explained by the liquid/liquid aqueous/organic extraction used which was followed by analysis

using GC-MS. Nicotine-N’-oxide is a highly polar and zwitterionic compound that is not readily

soluble in non-polar solvents such as dichloromethane.

β-Nicotyrine was not determined by many labs due to the extraction deficiency of the method

used or use of high method LOQ. β-Nicotyrine was detected in all EL and NP samples by labs

6 and 7 due to their low method LOQ (Appendix F and G).


8. Conclusions

It is apparent from the raw data graphs and the z-scores that in many cases labs obtained

different results for nicotine degradants and this is likely due to the use of different

methodologies. The standard deviations, calculated using robust statistical methods, were found

to be noticeably larger than the Horwitz standard deviation estimates. Standardizing the

analytical methods would likely improve the consistency of the results. The amount of nicotine

determined by all labs in all samples was found to be relatively consistent despite the use of

various extraction and analytical methods, but still more variable than would be expected with

a single standardized method. The determination of the seven nicotine degradants in one single

method requires the use of LC-MS/MS method. The use of isotopically labelled internal

standards for all nicotine degradants provided higher recoveries and more accurate quantitation

(compensate for ions enhancement/suppression and possibly for recovery loss and degradation

during extraction). Finally, it is highly recommended to use the nicotine-N’-oxides (cis and

trans; CAS #491-26-9) standard for the accurate quantitation of this analyte in samples.

9. Recommendations

Results from this study were discussed at the virtual TTPA and EVAP Sub-Group meetings

held on April 22 and 23, 2020. The TTPA Sub-Group recommended that a small group be

formed to develop a method that can be evaluated as a potential CORESTA Recommended

Method for the determination of nicotine degradants.


APPENDIX A: Study Protocol

CORESTA TOBACCO and TOBACCO PRODUCTS

ANALYTES SUB-GROUP and E-VAPOUR SUB-GROUP

Project Title: Proficiency Study for Nicotine and Nicotine Degradants in

Nicotine Pouches and E-Liquids

Type of Document: Proficiency Study Protocol

Date: October 30, 2019

Study Coordinator: Karl Wagner and Fadi Aldeek

Confidentiality Notice: All data submitted by participating laboratories will be coded

and kept confidential.


1. Introduction

At the CORESTA Tobacco and Tobacco Products Analytes Sub-Group (TTPA) meeting held

on October 6, 2019 in Hamburg, Germany, it was decided that a proficiency study for nicotine

and nicotine degradants in nicotine pouches and e-liquids would be conducted by TTPA and

the E-Vapour Sub-Group (EVAP) in 2020. For the purpose of this study, nicotine pouches are

defined as non-tobacco containing pouches with tobacco derived nicotine.

2. Objective

The objective of this study is to provide an assessment of laboratory proficiency for the

determination of nicotine and nicotine degradants in nicotine pouches and e-liquids. The final

output will be a technical report that includes z-scores and a presentation of the results at the

2020 spring TTPA and EVAP meetings.

3. Time schedule

Table 1: Study timeline

Date Activity

December 2019 Distribute the study protocol and data reporting sheet

January 6, 2020 Distribute study samples

January-February 2020 Laboratories to conduct the study

March 15, 2020 Study closes. Laboratories submit results by this date

March 2020 Statistical Analysis

April TTPA meeting Discuss results at Spring 2020 TTPA and EVAP meetings

4. Participating Laboratories:

The laboratories listed in Table 2 have kindly agreed to take part in the study. Other laboratories

are encouraged to participate and should notify Karl Wagner, Fadi Aldeek, Johan Lindholm

and John Bunch of their interest to participate.

Table 2: Participating Laboratories

Sample Types Company Country

both Altria Client Services US

both Swedish Match, SE Sweden

both R.J. Reynolds Tobacco Company US

both Enthalpy Analytical, Richmond US

both Labstat International Canada

both University of Kentucky US

both Global Laboratories US

both Imperial Tobacco- Reemtsma, DE Germany

both JTI OEKOLAB Austria

both KT&G, KR South Korea

both Eurofins SWE, SW Sweden

both Lauterbach & Associates, US US


Sample Types Company Country

both NIST US

both INCIENSA Costa Rica

both ITC Limited - Agri Business Division India

e-liquid only CNTC China National Tobacco Quality Supervision and

Test Center, CN China

e-liquid only CNTC Zhengzhou Tobacco Research Institute, CN China

both PMI Switzerland

both Essentra England

both Japan Tobacco INC Japan

5. Samples

The samples will be provided by the companies listed in Table 3. Swedish Match will provide

two fillers for nicotine pouch products (one artificially aged and one unaged), American Snuff

Co. will provide three fillers nicotine pouch products (two artificially aged and one unaged),

and Enthalpy Analytical will provide four e-liquid samples (three artificially aged/fortified and

one unaged/fortified). Shipping information should be sent to Karl Wagner, Fadi Aldeek, Johan

Lindholm and John Bunch. Unless analyzed within one week, samples shall be placed in

moisture barrier bags and stored at freezer conditions prior to analysis.

All remaining samples should be retained in sealed containers at under the specified

storage conditions as they may be required for a reanalysis.

Table 3: Samples

Sample Name

Description Supplier

NP-1 Nicotine pouch, homogeneous bulk sample without pouch paper Swedish Match

NP-2 Artificially aged nicotine pouch, homogeneous bulk sample

without pouch paper Swedish Match

NP-3 Nicotine pouch, homogeneous bulk sample without pouch paper American Snuff Co.


without pouch paper American Snuff Co.


without pouch paper American Snuff Co.

EL-1 E-liquid 1 Enthalpy Analytical

EL-2 Aged E-liquid 2 Enthalpy Analytical



6. Analysis

6.1 Analytes:

Each participating laboratory should report nicotine and nicotine degradants for

each sample. It is not necessary to report all analytes in order to participate in the

study. Be aware that the artificially aged samples may have significant levels of

certain degradants and may need to be diluted and reanalyzed.


6.2 Methods:

Each participating laboratory should follow their in-house analytical procedure for

the determination of nicotine and nicotine degradants.

Provide a synopsis of the method used to generate data in the Excel method specific

worksheet.

6.3 Replicates:

Conduct three (3) independent replicate analyses for each sample. The replicates

shall be determined from independently prepared sample aliquots.

Sample equilibration: Samples shall be removed from the freezer a minimum of 24

hours prior to analysis and placed in the refrigerator. Samples shall be removed

from the refrigerator a minimum of 2 hours prior to opening for analysis. The

samples shall not be opened during the time the samples are equilibrating to ambient

temperature.

6.4 Sample Handling Requirements:

Mix samples before removing aliquots for analysis.

6.5 Data Reporting:

The provided data reporting spreadsheet with the results of analysis and synopsis

of the method worksheets shall be sent by email to the study coordinators

K.A.Wagner and Fadi Aldeek. Report the data and provide a method synopsis in

the supplied reporting sheets:

• Data shall be reported to three decimal places.

• Please provide a synopsis of the method used to generate data.

6.6 Statistical Analysis

Z-Scores will be calculated for all samples.

7. Presentation of the Results

The results will be a presented for discussion at the spring 2020 TTPA and EVAP Sub-Group

meetings.

mailto:[email protected]


APPENDIX B: Data Tables

Data values in mg/g for nicotine and µg/g for nicotine degradants as-is

Lab Code

Sample ID Rep Nicotine Anabasine Anatabine β-Nicotyrine Cotinine Myosmine Nicotine-N’-Oxide

Nornicotine

1 NP-1 1 16.56 ND ND ND ND ND BLOQ ND



1 NP-2 1 13.56 ND ND ND ND BLOQ 7.30 BLOQ



1 NP-3 1 8.73 ND ND ND 1.91 1.91 28.20 BLOQ

1 NP-3 2 8.69 ND ND ND 1.95 1.73 28.30 BLOQ

1 NP-3 3 8.72 ND ND ND 1.85 2.07 28.20 BLOQ

1 NP-4 1 8.39 ND ND ND 3.21 2.25 383.00 3.17

1 NP-4 2 8.35 ND ND ND 3.26 1.93 392.00 2.76

1 NP-4 3 8.28 ND ND ND 3.16 2.10 375.00 2.92

1 NP-5 1 8.47 ND ND ND 7.91 3.22 881.00 4.12

1 NP-5 2 8.43 ND ND ND 7.19 3.29 835.00 4.74

1 NP-5 3 8.54 ND ND ND 7.42 3.34 846.00 4.50

1 EL-1 1 12.72 41.70 65.40 BLOQ 45.40 48.30 48.40 29.60

1 EL-1 2 12.84 41.50 67.00 BLOQ 42.10 48.10 48.00 28.40

1 EL-1 3 12.91 43.90 67.40 BLOQ 42.40 50.10 49.20 30.50

1 EL-2 1 13.21 18.90 43.70 BLOQ 17.60 24.30 14.30 3.94

1 EL-2 2 13.32 18.90 41.60 BLOQ 17.20 29.00 14.30 4.20



Lab Code


Nornicotine

1 EL-2 3 13.23 19.00 44.40 BLOQ 16.50 27.90 14.80 4.69

1 EL-3 1 13.51 87.60 119.00 BLOQ 87.70 89.70 119.00 97.40

1 EL-3 2 13.38 86.30 116.00 BLOQ 92.20 87.90 122.00 96.80

1 EL-3 3 13.37 89.10 111.00 BLOQ 90.90 77.20 120.00 99.30

1 EL-4 1 37.11 1.50 1.13 BLOQ 14.10 16.80 180.00 6.95

1 EL-4 2 36.74 1.55 1.11 BLOQ 14.10 17.70 171.00 7.30

1 EL-4 3 36.34 1.55 1.21 BLOQ 14.40 18.00 174.00 7.26

2 NP-1 1 13.05 BLOQ BLOQ NA NA BLOQ NA BLOQ









2 NP-4 1 8.45 BLOQ BLOQ NA NA BLOQ NA 46.38








Lab Code


Nornicotine

2 EL-1 1 NA NA NA NA NA NA NA NA












3 NP-1 1 16.53 NA NA NA NA NA NA NA












Lab Code


Nornicotine






3 EL-1 1 12.76 NA NA NA NA NA NA NA












4 NP-1 1 16.83 ND ND ND BLOQ BLOQ 0.79 BLOQ



4 NP-2 1 14.29 ND ND BLOQ 0.26 0.51 7.76 1.02

4 NP-2 2 13.67 ND ND BLOQ 0.26 0.50 8.35 0.87



Lab Code


Nornicotine

4 NP-2 3 13.99 ND ND BLOQ 0.26 0.53 8.17 0.89

4 NP-3 1 8.87 ND ND BLOQ 2.27 1.55 32.10 0.82

4 NP-3 2 8.88 ND ND BLOQ 2.40 1.62 33.10 0.94

4 NP-3 3 8.85 ND ND BLOQ 2.18 1.88 31.90 0.88

4 NP-4 1 8.52 ND ND BLOQ 3.71 1.91 368.00 2.72

4 NP-4 2 8.50 ND ND BLOQ 4.09 1.98 369.00 2.74

4 NP-4 3 8.54 ND ND BLOQ 3.71 1.99 373.00 2.54

4 NP-5 1 8.55 ND ND 0.40 9.35 3.10 823.00 3.86

4 NP-5 2 8.60 ND ND 0.35 8.24 2.78 793.00 3.51

4 NP-5 3 8.57 ND ND 0.37 8.22 3.03 842.00 3.63

4 EL-1 1 12.59 38.31 84.44 BLOQ 47.04 42.13 47.04 34.03

4 EL-1 2 12.57 39.96 90.78 BLOQ 49.95 44.57 48.80 34.97

4 EL-1 3 12.59 36.31 82.58 BLOQ 47.04 41.20 46.35 33.05

4 EL-2 1 13.13 17.49 58.16 BLOQ 19.90 26.09 11.98 3.19

4 EL-2 2 13.16 17.28 57.12 BLOQ 19.79 25.69 11.82 3.04

4 EL-2 3 13.14 17.66 56.59 BLOQ 20.25 25.20 12.64 2.90

4 EL-3 1 12.91 80.53 144.38 ND 105.62 78.99 121.20 110.87

4 EL-3 2 12.90 80.32 143.94 BLOQ 105.57 79.51 123.81 106.92

4 EL-3 3 12.93 80.25 141.12 BLOQ 107.94 79.40 121.83 113.33

4 EL-4 1 36.76 BLOQ BLOQ BLOQ 16.39 17.09 178.88 7.80

4 EL-4 2 36.78 BLOQ BLOQ BLOQ 17.26 18.28 182.09 8.02

4 EL-4 3 36.79 BLOQ BLOQ BLOQ 15.44 16.81 170.01 6.98



Lab Code


Nornicotine

5 NP-1 1 16.40 ND ND NA ND 3.48 NA 22.89

5 NP-1 2 15.59 ND ND NA ND 3.47 NA 21.95

5 NP-1 3 14.38 ND ND NA ND 3.01 NA 21.55

5 NP-2 1 11.08 ND ND NA ND 2.62 NA 19.79

5 NP-2 2 11.09 ND ND NA ND 2.65 NA 20.20

5 NP-2 3 11.21 ND ND NA ND 2.76 NA 19.09

5 NP-3 1 9.13 ND ND NA BLOQ 3.73 NA 17.23

5 NP-3 2 9.19 ND ND NA BLOQ 3.88 NA 15.68

5 NP-3 3 9.14 ND ND NA BLOQ 3.83 NA 16.20

5 NP-4 1 8.76 ND ND NA BLOQ 3.93 NA 16.91

5 NP-4 2 8.72 ND ND NA BLOQ 4.03 NA 17.86

5 NP-4 3 8.74 ND ND NA BLOQ 4.11 NA 17.25

5 NP-5 1 8.91 ND ND NA 10.25 5.06 NA 23.37

5 NP-5 2 8.86 ND ND NA 10.14 5.28 NA 23.30

5 NP-5 3 8.89 ND ND NA 10.02 5.17 NA 23.66

5 EL-1 1 13.65 52.28 145.71 NA 54.05 45.49 NA 91.62

5 EL-1 2 13.58 52.20 147.47 NA 53.47 45.20 NA 89.58

5 EL-1 3 13.77 52.50 146.00 NA 54.08 46.29 NA 91.48

5 EL-2 1 13.72 26.68 101.59 NA BLOQ 26.86 NA 44.26

5 EL-2 2 13.88 26.80 102.54 NA BLOQ 27.45 NA 44.89

5 EL-2 3 13.92 26.76 102.92 NA BLOQ 26.93 NA 45.89

5 EL-3 1 14.02 95.83 229.19 NA 116.14 79.48 NA 186.32



Lab Code


Nornicotine

5 EL-3 2 13.95 96.72 234.15 NA 118.58 78.40 NA 188.42

5 EL-3 3 13.84 96.36 229.73 NA 118.23 78.33 NA 184.99

5 EL-4 1 38.93 5.96 ND NA BLOQ 27.14 NA 72.03

5 EL-4 2 38.18 5.63 ND NA BLOQ 26.60 NA 71.07

5 EL-4 3 38.29 5.84 ND NA BLOQ 26.49 NA 73.79

6 NP-1 1 16.93 ND ND 0.01 0.19 2.90 1.16 BLOQ

6 NP-1 2 16.33 ND ND 0.01 0.25 BLOQ 1.36 BLOQ

6 NP-1 3 16.22 ND ND 0.02 0.18 BLOQ 1.24 BLOQ

6 NP-2 1 13.70 ND ND 0.10 0.46 BLOQ 9.53 BLOQ

6 NP-2 2 13.03 ND ND 0.10 0.46 BLOQ 9.48 BLOQ

6 NP-2 3 13.49 ND ND 0.11 0.42 BLOQ 9.76 BLOQ

6 NP-3 1 9.75 ND ND 0.06 2.86 3.70 43.46 BLOQ

6 NP-3 2 9.18 ND ND 0.06 2.52 3.55 39.05 BLOQ

6 NP-3 3 9.03 ND ND 0.06 2.65 3.54 40.95 BLOQ

6 NP-4 1 8.82 ND ND 0.15 4.04 3.33 538.63 BLOQ

6 NP-4 2 8.84 ND ND 0.16 4.19 4.35 536.72 BLOQ

6 NP-4 3 8.93 ND ND 0.13 4.15 4.58 538.65 BLOQ

6 NP-5 1 9.33 ND ND 0.36 8.89 5.58 1250.64 BLOQ

6 NP-5 2 9.44 ND ND 0.34 9.25 6.00 1203.26 BLOQ

6 NP-5 3 9.23 ND ND 0.36 9.18 5.66 1256.60 BLOQ

6 EL-1 1 12.89 44.18 95.59 1.61 49.54 50.68 66.59 BLOQ

6 EL-1 2 12.85 47.28 96.64 1.67 51.94 51.68 68.40 93.36



Lab Code


Nornicotine

6 EL-1 3 12.92 46.61 100.02 1.60 51.30 51.61 67.21 98.35

6 EL-2 1 13.16 27.69 68.25 1.80 21.26 29.73 22.92 BLOQ

6 EL-2 2 13.07 27.04 66.31 1.70 20.82 29.84 21.82 BLOQ

6 EL-2 3 13.11 25.44 67.59 1.87 20.09 28.79 24.31 BLOQ

6 EL-3 1 13.27 83.35 159.29 1.61 104.62 89.65 155.37 165.01

6 EL-3 2 13.26 90.53 162.77 1.63 105.79 91.07 159.37 163.99

6 EL-3 3 13.15 87.96 160.99 1.75 104.24 90.15 156.64 166.67

6 EL-4 1 35.66 16.42 ND 1.07 17.22 22.27 223.95 BLOQ

6 EL-4 2 36.28 BLOQ ND 1.09 17.88 21.32 222.37 BLOQ

6 EL-4 3 36.54 BLOQ ND 1.12 17.35 21.65 224.04 BLOQ

7 NP-1 1 14.67 ND ND ND ND ND 22.23 12.39

7 NP-1 2 15.02 ND ND ND ND ND 15.24 12.44

7 NP-1 3 15.23 ND ND ND BLOQ ND 14.10 16.60

7 NP-2 1 13.48 ND ND 0.11 BLOQ 0.54 34.48 11.61

7 NP-2 2 12.72 ND ND 0.13 BLOQ 0.83 23.26 10.61

7 NP-2 3 13.42 ND ND BLOQ BLOQ 0.53 28.40 12.50

7 NP-3 1 8.47 ND ND BLOQ 2.78 1.85 64.02 10.77

7 NP-3 2 8.85 ND ND BLOQ 2.57 1.76 67.71 8.96

7 NP-3 3 8.81 ND ND BLOQ 2.62 1.89 72.60 10.10

7 NP-4 1 8.20 ND ND 0.12 4.06 2.07 747.89 10.49

7 NP-4 2 7.69 ND ND 0.13 4.04 2.13 765.24 11.02

7 NP-4 3 8.13 ND ND BLOQ 3.82 2.06 898.50 9.90



Lab Code


Nornicotine

7 NP-5 1 7.02 ND ND 0.31 9.05 3.06 1474.08 12.45

7 NP-5 2 8.35 ND ND 0.30 8.96 2.95 1432.24 12.04

7 NP-5 3 8.45 ND ND 0.29 7.62 3.08 1908.20 11.80

7 EL-1 1 11.86 47.48 103.19 1.34 57.51 50.62 136.03 55.82

7 EL-1 2 11.72 37.70 80.46 1.35 45.42 39.44 104.29 41.16

7 EL-1 3 10.56 37.61 80.35 1.41 49.37 42.21 107.00 41.12

7 EL-2 1 11.32 18.79 56.31 1.49 18.91 25.83 39.73 16.43

7 EL-2 2 12.32 14.98 49.61 1.41 17.93 22.32 37.97 14.81

7 EL-2 3 11.45 17.03 50.44 1.49 18.80 22.85 33.80 14.21

7 EL-3 1 12.05 86.74 144.27 1.49 109.30 74.08 219.98 118.72

7 EL-3 2 11.77 91.96 152.61 1.45 110.38 78.33 243.45 123.95

7 EL-3 3 12.21 74.80 141.90 1.43 103.00 66.61 253.40 83.20

7 EL-4 1 37.23 1.61 1.12 0.85 14.48 15.55 378.48 45.34

7 EL-4 2 34.59 1.49 1.20 0.82 15.66 15.36 391.55 44.95

7 EL-4 3 36.56 1.39 1.30 0.86 15.70 16.50 358.20 42.14

8 NP-1 1 17.96 ND ND NA ND ND NA 5.23

8 NP-1 2 17.46 ND ND NA ND ND NA 3.47

8 NP-1 3 17.70 ND ND NA ND ND NA 4.10

8 NP-2 1 13.85 ND ND NA ND ND NA 4.66

8 NP-2 2 13.76 ND ND NA ND ND NA 5.12

8 NP-2 3 13.46 ND ND NA ND ND NA 4.49

8 NP-3 1 9.46 ND ND NA ND ND NA 5.75



Lab Code


Nornicotine

8 NP-3 2 9.31 ND ND NA ND ND NA 5.47

8 NP-3 3 9.29 ND ND NA ND ND NA 4.90

8 NP-4 1 9.46 ND ND NA ND ND NA 9.06

8 NP-4 2 9.63 ND ND NA ND ND NA 7.70

8 NP-4 3 9.50 ND ND NA ND ND NA 8.00

8 NP-5 1 9.78 ND ND NA ND ND NA 16.70

8 NP-5 2 9.83 ND ND NA ND ND NA 14.17

8 NP-5 3 9.76 ND ND NA ND ND NA 15.06

8 EL-1 1 13.51 34.79 5.23 NA 1.69 6.71 NA 48.17

8 EL-1 2 13.33 35.17 6.81 NA 3.88 7.13 NA 44.52

8 EL-1 3 12.97 33.55 9.18 NA 1.84 8.31 NA 46.56

8 EL-2 1 13.26 15.24 ND NA ND ND NA 8.88

8 EL-2 2 13.25 15.25 ND NA ND ND NA 8.87

8 EL-2 3 13.05 16.03 ND NA ND ND NA 9.63

8 EL-3 1 13.22 81.18 50.84 NA 39.81 39.55 NA 159.61

8 EL-3 2 13.13 80.36 52.34 NA 55.58 40.81 NA 156.98

8 EL-3 3 13.26 81.43 45.30 NA 38.27 34.34 NA 164.42

8 EL-4 1 35.49 ND ND NA ND ND NA 17.67

8 EL-4 2 35.36 ND ND NA ND ND NA 17.21

8 EL-4 3 34.76 ND ND NA ND ND NA 15.76

9 NP-1 1 13.85 ND ND ND ND ND 1.55 BLOQ




Lab Code


Nornicotine


9 NP-2 1 11.00 ND ND ND BLOQ BLOQ 9.60 1.15



9 NP-3 1 7.70 ND ND ND 2.50 2.05 70.25 1.45

9 NP-3 2 7.76 ND ND ND 2.50 2.10 65.90 1.50

9 NP-3 3 8.29 ND ND ND 2.50 2.20 68.20 1.40

9 NP-4 1 7.32 ND ND ND 4.00 2.50 521.90 3.75

9 NP-4 2 7.58 ND ND ND 4.05 2.45 509.45 3.80

9 NP-4 3 7.69 ND ND ND 3.95 2.25 516.25 3.35

9 NP-5 1 7.92 ND ND ND 9.00 3.85 1162.00 5.00

9 NP-5 2 8.14 ND ND ND 9.30 3.60 1134.95 5.75

9 NP-5 3 8.23 ND ND ND 9.25 3.65 1152.50 5.05

9 EL-1 1 11.26 39.85 101.50 2.05 43.80 47.65 56.65 31.95

9 EL-1 2 11.67 33.70 104.65 2.05 46.45 47.95 56.55 35.35

9 EL-1 3 11.79 33.90 103.35 2.15 44.80 45.00 57.65 28.75

9 EL-2 1 11.31 18.30 64.95 2.20 18.20 28.05 17.40 2.95

9 EL-2 2 11.92 14.70 65.55 BLOQ 18.85 25.60 19.35 2.85

9 EL-2 3 10.80 15.40 65.70 2.55 17.85 25.30 20.05 2.60

9 EL-3 1 12.86 83.80 170.55 2.05 95.95 79.80 127.25 115.15

9 EL-3 2 12.30 70.15 169.10 BLOQ 97.75 84.60 134.85 112.00

9 EL-3 3 12.30 70.75 175.00 2.35 101.10 81.65 133.70 99.90



Lab Code


Nornicotine

9 EL-4 1 32.01 1.80 1.75 BLOQ 14.45 17.70 198.25 8.70

9 EL-4 2 31.04 1.20 1.75 BLOQ 14.25 17.90 194.60 8.95

9 EL-4 3 34.43 1.30 1.70 BLOQ 14.75 18.35 198.75 8.60


10 NP-1 2 16.82 ND ND ND ND ND ND ND





10 NP-3 1 8.95 ND ND ND BLOQ BLOQ 26.94 ND









10 EL-1 1 13.13 45.47 82.61 BLOQ 48.37 46.10 54.77 27.24

10 EL-1 2 12.81 43.34 80.37 BLOQ 45.72 44.09 51.85 25.04

10 EL-1 3 12.85 42.91 78.58 BLOQ 45.11 43.11 50.68 24.76

10 EL-2 1 13.29 18.46 52.23 BLOQ 18.18 25.03 20.74 ND



Lab Code


Nornicotine

10 EL-2 2 13.35 19.65 53.49 BLOQ 19.01 25.84 21.26 ND

10 EL-2 3 13.37 18.62 53.48 BLOQ 18.25 25.30 21.36 ND

10 EL-3 1 13.24 88.77 142.25 BLOQ 100.47 79.62 143.35 96.76

10 EL-3 2 13.19 92.43 140.95 BLOQ 101.80 79.87 141.54 95.54

10 EL-3 3 13.23 88.76 142.21 BLOQ 101.56 79.67 141.31 94.37

10 EL-4 1 37.94 ND ND BLOQ 14.16 17.17 177.09 BLOQ

10 EL-4 2 37.37 ND ND BLOQ 14.36 17.49 174.16 BLOQ

10 EL-4 3 37.46 ND ND BLOQ 14.53 17.25 175.21 BLOQ

11 NP-1 1 14.61 ND ND ND ND ND 1.00 ND

11 NP-1 2 15.37 ND ND ND ND ND 0.99 ND

11 NP-1 3 14.55 ND ND ND ND ND 1.01 ND

11 NP-2 1 14.60 ND ND ND ND 0.59 7.19 0.92

11 NP-2 2 13.42 ND ND ND ND 0.59 7.13 0.92

11 NP-2 3 14.68 ND ND ND ND 0.56 7.35 0.96

11 NP-3 1 8.24 ND ND ND 2.29 1.79 87.14 1.36

11 NP-3 2 9.06 ND ND ND 2.29 1.90 87.91 1.36

11 NP-3 3 8.55 ND ND ND 2.40 1.79 91.17 1.43

11 NP-4 1 8.34 ND ND BLOQ 3.75 2.22 411.65 2.68

11 NP-4 2 8.42 ND ND BLOQ 3.96 2.03 441.82 2.68

11 NP-4 3 8.50 ND ND 0.50 4.01 2.17 379.07 2.79

11 NP-5 1 9.07 ND ND 1.06 8.82 3.19 896.87 3.11

11 NP-5 2 8.96 ND ND 1.07 8.34 3.04 899.29 3.01



Lab Code


Nornicotine

11 NP-5 3 9.25 ND ND 1.04 8.38 3.08 909.64 3.06

11 EL-1 1 37.28 ND ND ND 13.96 17.83 175.65 8.63

11 EL-1 2 39.11 ND ND ND 14.94 18.00 172.44 8.93

11 EL-1 3 39.90 ND ND ND 13.97 17.84 171.34 9.08

11 EL-2 1 3.33 39.72 80.98 BLOQ 45.15 45.76 56.56 20.44

11 EL-2 2 3.75 40.94 84.05 BLOQ 46.52 42.62 57.46 20.46

11 EL-2 3 4.05 42.36 83.91 BLOQ 46.39 48.47 58.00 20.89

11 EL-3 1 3.94 17.75 49.69 BLOQ 17.92 25.00 20.11 ND

11 EL-3 2 4.71 18.39 51.60 BLOQ 18.56 26.08 20.81 ND

11 EL-3 3 4.34 18.53 51.70 BLOQ 18.68 25.65 21.13 ND

11 EL-4 1 5.96 90.19 151.87 6.54 107.20 93.10 159.68 84.09

11 EL-4 2 4.94 83.91 143.34 5.67 101.17 83.73 148.58 82.60

11 EL-4 3 5.24 87.51 144.10 5.61 104.72 81.84 153.03 85.55

12 NP-1 1 17.46 ND ND 0.91 1.05 BLOQ NA 0.91

12 NP-1 2 19.72 ND ND 1.09 0.84 BLOQ NA 0.85

12 NP-1 3 18.69 ND ND 1.31 0.85 BLOQ NA 0.86

12 NP-2 1 NA NA NA NA NA NA NA NA



12 NP-3 1 9.27 ND ND ND 1.53 1.72 NA 1.14

12 NP-3 2 9.37 ND ND ND 1.52 1.86 NA 1.29

12 NP-3 3 9.20 ND ND ND 1.28 1.87 NA 1.21



Lab Code


Nornicotine

12 NP-4 1 9.06 ND ND BLOQ 2.27 2.28 NA 3.05

12 NP-4 2 8.77 ND ND BLOQ 2.21 2.27 NA 2.96

12 NP-4 3 11.61 ND ND BLOQ 2.34 2.33 NA 2.90

12 NP-5 1 9.74 ND ND 1.02 5.09 3.75 NA 4.47

12 NP-5 2 10.25 ND ND 1.20 5.45 3.96 NA 4.62

12 NP-5 3 9.75 ND ND 1.44 5.53 3.96 NA 5.14



















Lab Code


Nornicotine

























Lab Code


Nornicotine

























Lab Code


Nornicotine

























Lab Code


Nornicotine












16 NP-1 1 14.28 ND ND ND ND ND NA ND

16 NP-1 2 14.80 ND ND ND ND BLOQ NA ND

16 NP-1 3 15.66 ND ND ND ND BLOQ NA BLOQ


16 NP-2 2 11.96 ND ND ND ND BLOQ NA ND


16 NP-3 1 9.93 ND ND ND BLOQ 3.28 NA ND

16 NP-3 2 8.70 ND ND ND BLOQ 3.49 NA BLOQ






Lab Code


Nornicotine





16 EL-1 1 11.12 45.14 91.71 ND 19.35 58.31 NA 25.90

16 EL-1 2 12.31 45.42 93.36 ND 20.02 56.55 NA 26.57

16 EL-1 3 12.70 46.27 92.79 ND 19.34 58.43 NA 26.77

16 EL-2 1 12.62 22.27 60.54 ND BLOQ 33.45 NA BLOQ

16 EL-2 2 12.76 18.28 59.08 ND BLOQ 31.63 NA BLOQ

16 EL-2 3 11.57 19.71 60.21 ND BLOQ 32.96 NA BLOQ

16 EL-3 1 12.73 97.67 159.04 BLOQ 43.79 105.56 NA 92.09

16 EL-3 2 12.27 93.61 153.86 BLOQ 42.00 100.20 NA 90.26

16 EL-3 3 12.51 93.57 154.97 BLOQ 42.13 99.82 NA 90.71

16 EL-4 1 24.45 ND ND BLOQ BLOQ 22.61 NA BLOQ



17 NP-1 1 23.61 ND BLOQ BLOQ ND BLOQ 2.20 8.74








Lab Code


Nornicotine

17 NP-3 1 13.64 ND BLOQ BLOQ 3.41 2.28 37.13 10.45

17 NP-3 2 13.29 ND BLOQ 1.59 1.88 1.50 28.18 4.78

17 NP-3 3 16.24 ND BLOQ ND 1.80 1.54 28.43 2.11

17 NP-4 1 12.64 ND BLOQ ND 3.80 2.01 640.03 8.56

17 NP-4 2 12.61 ND BLOQ ND 3.81 2.00 653.47 9.21

17 NP-4 3 12.74 ND BLOQ ND 3.45 1.86 604.51 7.99




17 EL-1 1 13.79 52.73 114.25 1.16 36.63 36.00 97.20 39.53

17 EL-1 2 13.38 57.24 131.20 1.21 39.48 39.27 103.68 42.57

17 EL-1 3 14.74 49.81 99.87 1.08 34.00 33.62 89.24 35.76

17 EL-2 1 16.76 12.87 98.45 1.01 14.60 18.59 51.57 6.99

17 EL-2 2 20.22 19.50 179.36 1.62 23.00 29.63 77.45 11.24

17 EL-2 3 18.44 19.75 190.65 1.81 24.14 31.10 70.84 10.77

17 EL-3 1 23.22 89.52 62.43 0.67 61.49 64.01 106.67 127.26

17 EL-3 2 29.11 112.24 80.41 0.76 73.50 77.88 124.17 157.17

17 EL-3 3 26.23 87.08 57.00 0.63 61.79 64.29 113.14 133.75

17 EL-4 1 17.17 20.47 55.78 1.10 30.82 29.66 242.33 49.05

17 EL-4 2 18.71 23.47 76.70 1.55 36.36 40.57 287.93 60.94

17 EL-4 3 20.35 19.24 51.44 0.97 29.12 28.41 215.99 45.69




Lab Code


Nornicotine















18 EL-1 1 13.68 31.74 67.80 5.59 62.80 39.19 ND 15.09

18 EL-1 2 14.32 28.30 61.28 6.22 60.36 39.93 ND 15.70

18 EL-1 3 13.75 28.96 66.55 5.33 67.05 38.97 ND 14.64

18 EL-2 1 14.35 15.75 39.26 4.60 14.36 21.96 ND 3.56

18 EL-2 2 14.23 16.71 38.06 5.08 15.26 22.60 ND 3.91

18 EL-2 3 13.90 16.99 42.20 4.07 16.89 22.49 ND 4.24

18 EL-3 1 14.33 66.73 128.65 6.05 138.43 74.64 ND 72.28

18 EL-3 2 14.56 66.61 126.92 4.58 138.62 74.43 ND 75.40



Lab Code


Nornicotine

18 EL-3 3 14.75 63.38 119.59 4.37 126.32 71.01 ND 77.47

18 EL-4 1 37.14 6.73 3.78 11.91 13.95 17.71 BLOQ 14.45

18 EL-4 2 37.43 6.59 3.91 9.46 14.70 18.00 BLOQ 15.21

18 EL-4 3 37.64 7.05 3.84 7.43 12.34 18.16 BLOQ 14.27

19 NP-1 1 21.08 ND ND ND ND ND ND 4.68

19 NP-1 2 22.40 ND ND ND ND ND ND 4.59

19 NP-1 3 24.32 ND ND ND ND ND ND 4.94

19 NP-2 1 14.91 ND ND ND ND ND BLOQ 4.86



19 NP-3 1 11.30 ND ND ND 1.74 ND 41.80 4.17

19 NP-3 2 10.03 ND ND ND 1.80 ND 42.78 4.12

19 NP-3 3 11.65 ND ND ND 1.74 ND 41.40 BLOQ

19 NP-4 1 10.28 ND ND ND 2.99 BLOQ 540.46 5.24

19 NP-4 2 10.04 ND ND ND 3.36 BLOQ 549.00 4.86

19 NP-4 3 10.58 ND ND ND 3.30 BLOQ 540.67 4.86

19 NP-5 1 10.83 ND ND ND 7.87 BLOQ 1370.87 6.28

19 NP-5 2 10.37 ND ND ND 7.65 BLOQ 1317.47 6.22

19 NP-5 3 11.05 ND ND ND 8.06 BLOQ 1377.44 6.22

19 EL-1 1 16.39 37.69 77.61 BLOQ 45.39 46.27 58.15 31.92

19 EL-1 2 16.42 38.12 75.90 BLOQ 46.21 45.50 59.47 30.90

19 EL-1 3 16.76 38.10 76.94 BLOQ 47.02 45.13 59.02 31.36



Lab Code


Nornicotine

19 EL-2 1 15.90 15.94 49.86 BLOQ 18.23 24.37 17.50 4.99

19 EL-2 2 17.00 16.79 49.85 BLOQ 19.10 26.78 17.64 8.12

19 EL-2 3 16.97 16.63 49.70 BLOQ 19.02 26.52 17.83 7.89

19 EL-3 1 17.06 85.00 132.72 BLOQ 103.22 81.77 152.89 102.34

19 EL-3 2 16.87 81.82 136.11 BLOQ 101.75 84.24 145.87 101.96

19 EL-3 3 17.13 82.10 133.17 BLOQ 102.00 81.95 150.27 102.05

19 EL-4 1 45.62 ND ND BLOQ 14.83 17.03 235.23 10.90

19 EL-4 2 43.77 ND ND BLOQ 14.57 16.91 231.10 11.95

19 EL-4 3 45.07 ND ND BLOQ 14.22 16.16 230.62 12.13

1. “NA” signifies the laboratory did not submit data for this sample/element.

2. “ND” signifies that one or more of the reported values was between the limit of detection and the limit of quantitation

3. “BLOQ” signifies the data were below the limit of quantitation.


APPENDIX C: Raw Data Plots









APPENDIX D: Graphs of z-scores




Note: Two values for Lab 11 are outside the graph. The scale was truncated to allow easier visualization of the remaining points.





APPENDIX E: Summary of Method Synopses for Nicotine Analysis

Lab Code

Sample Weight (g)

Extraction Volume (mL)

Extraction Time (min)

Solvent Dilution Factor

MLOQ (ug/g)

Analysis

1 1 50 120 MTBE 50 2000 GC-FID

2 0.15 4.1 30 MeOH/DCM (1:3 v/v) 1 90 GC-MS

3 1 100 240 Methanolic-KOH 400 5 LC-MS

4 1 50 120 MTBE 50 3000 GC-FID

5 0.5 25 960 MTBE ND 1580 GC-MS

6 0.25 40 30 MeOH 1 240 GC-MS

7 0.1 10 5 10 % Acetonitrile 1 1000 LC-MS/MS

8 0.1 5 120 MeOH 1 2000 GC-MS/MS

9 0.5 25 30 2N NaOH+MeOH 200 100 GC-FID

10 1 50 120 MTBE ND ND GC-FID

11 1 20 30 70:30 MeOH:H2O 50 0.5 LC-MS/MS

12 0.25 40 30 MeOH 1 11.8 GC-MS

13 0.25 40 30 MeOH 1 6 GC-MS

14 0.1 10 10 IPA ND ND GC-FID

15 1 15 10 Acetonitrile 5 7.5 LC-PDA

16 1 50 60 MTBE 1 1100 GC-MS

17 0.3 10 30 Isopropanol 1 1.4 LC-MS/MS

18 0.3 10 40 DCM 1 330 GC-MS

19 0.1 20 60 100 mM Ammonium acetate 1000 5070 LC-MS/MS

*ND: Data were not provided by the lab.


APPENDIX F: Summary of Method Synopses for Nicotine Degradants Analysis in NP Samples

Lab Code

Sample Weight (g)

Extraction Volume (mL)


Solvent MLOQ (ug/g)

Analysis

1 1 50 40 100 mM Ammonium Formate pH 3.0 1-to-2 LC-MS/MS

2 0.15 4.1 30 MeOH/DCM (1:3 v/v) 6-to-30 GC-MS

4 1 10 30 70:30 MeOH:H2O 0.25 LC-MS/MS

5 0.5 25 960 MTBE 0.3-to-2 GC-MS

6 0.25 20-to-40 30 MeOH 0.02-to-93 LC-MS/MS &

GC-MS

7 0.1 10 5 10 % Acetonitrile 0.1-to-1.5 LC-MS/MS

8 0.1 5 120 MeOH 7-to-50 GC-MS


10 0.05 5 until mixed 70:30 MeOH:H2O 10 LC-MS/MS

11 1 20 30 70:30 MeOH:H2O 0.5 LC-MS/MS

12 1 40 30 MeOH 0.18-to-0.5 GC-MS

16 1 50 60 MTBE 1.4-to-15 GC-MS

17 0.3 10 30 Isopropanol 1.4 LC-MS/MS

18 0.3 10 40 DCM 0.3-to-2 GC-MS

19 0.1 20 60 100 mM Ammonium acetate 1.5-to-11 LC-MS/MS

Note: Labs 3, 13, 14, and 15 did not participate in the nicotine degradants analysis


APPENDIX G: Summary of Method Synopses for Nicotine Degradants Analysis in EL Samples

Lab Code

Sample Weight (g)

Extraction

Volume (mL)


Solvent MLOQ (µg/g)

Analysis


2 0.15 4.1 30 MeOH/DCM (1:3 v/v) 6-to-30 GC-MS

4 0.1 10 30 70:30 MeOH:H2O 2.5 LC-MS/MS

5 0.1 25 960 MTBE 1.6-to-10.4 GC-MS

6 0.25 20-to-40 30 MeOH 0.02-to-93 LC-MS/MS & GC-MS

7 0.1 10 5 10 % Acetonitrile 0.1-to-1.5 LC-MS/MS

8 0.1 5 120 MeOH 7-to-50 GC-MS


10 0.05 5 until mixed 70:30 MeOH:H2O 10 LC-MS/MS

11 0.1 20 30 70:30 MeOH:H2O 5 LC-MS/MS

16 0.4 50 60 MTBE 3.5-to-37.5 GC-MS

17 0.3 10 30 Isopropanol 1.4 LC/MS/MS

18 0.3 10 40 DCM 0.3-to-2 GC-MS

19 0.1 20 60 100 mM Ammonium acetate 1.5-to-11 LC/MS/MS

Note: Labs 3, 13, 14, and 15 did not participate in the nicotine degradants analysis


APPENDIX H: Summary of Internal Standards used for each Analyte

Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Lab 7 Lab 8

Nicotine Quinoline Quinoline Nicotine-d4 Quinoline Methylquinoline Quinoline Nicotine-d3 Quinoline

Anabasine Anabasine-d4 4,4'-Dipyridyl NA Anabasine-d4 Methylquinoline Methylquinoline Anabasine-d4 Quinoline

Anatabine Anatabine-d4 4,4'-Dipyridyl NA Myosmine-d4 Methylquinoline Methylquinoline Anatabine-d4 Quinoline

β-Nicotyrine β-Nicotyrine-d3 NA NA Myosmine-d4 Methylquinoline β-Nicotyrine-d3 β-Nicotyrine-d3 NA

Cotinine Cotinine-d3 NA NA Cotinine-d3 Methylquinoline Cotinine-d3 Cotinine-d3 Quinoline

Myosmine Myosmine-d4 4,4'-Dipyridyl NA Myosmine-d4 Methylquinoline Methylquinoline Myosmine-d4 Quinoline

Nicotine-N’-Oxide

Nicotine-N-Oxide-d3 NA NA Cotinine-d3 Methylquinoline Nicotine-N’-Oxide-d3 Nicotine-N’-Oxide-d3 NA

Nornicotine Nornicotine-d4 4,4'-Dipyridyl NA Nornicotine-

d4 Methylquinoline Methylquinoline Nornicotine-d4 Quinoline

Lab 9 Lab 10 Lab 11 Lab 12 Lab 13 Lab 14 Lab 15 Lab 16

Nicotine NA NA ND Quinoline Nicotine-D4 n-Heptadecane Caffeine Quinoline

Anabasine NA Anabasine-d4 ND Quinoline NA NA NA 4,4’-Dipyridyl

Anatabine Anatabine-d4 Myosmine-d4 ND Quinoline NA NA NA 4,4’-Dipyridyl

β-Nicotyrine NA Myosmine-d4 ND Quinoline NA NA NA 4,4’-Dipyridyl

Cotinine Cotinine-d3 Cotinine-d3 ND Quinoline NA NA NA 4,4’-Dipyridyl

Myosmine Myosmine-d4 Myosmine-d4 ND Quinoline NA NA NA 4,4’-Dipyridyl

Nicotine-N’-Oxide

NA Myosmine-d4 ND Quinoline NA NA NA NA

Nornicotine Nornicotine-d4 Nornicotine-d4 ND Quinoline NA NA NA 4,4’-Dipyridyl

Lab 17 Lab 18 Lab 19

Nicotine Quinoline Quinoline Nicotine-d3

Anabasine Quinoline 2,2’-Dipyridyl-d8 Anabasine-d4

Anatabine Quinoline 2,2’-Dipyridyl-d8 Anatabine-d4

β-Nicotyrine Quinoline 2,2’-Dipyridyl-d8 β-Nicotyrine-d3

Cotinine Quinoline 2,2’-Dipyridyl-d8 Cotinine-d3

Myosmine Quinoline 2,2’-Dipyridyl-d8 Myosmine-d4

Nicotine-N’-Oxide Quinoline 2,2’-Dipyridyl-d8 Nicotine-N-Oxide-d3

Nornicotine Quinoline Nornicotine-d4 Nornicotine-d4

1. “NA” signifies the laboratory did not submit data for this sample/element.

2. “ND” signifies that the lab did report the internal standard used for the analyte if interest

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