chemical analysis in the regulation of tobacco products · regional trends for selected hphcs in...
TRANSCRIPT
Chris Wright PhD, Derek Mariner PhD, Mark Williams MBA & Chris Proctor PhD
British American Tobacco Group Research & Development, Southampton UK
1
68th Tobacco Science Research Conference, Charlottesville VA, 28 Sept – 1 Oct 2014
Chemical analysis in the regulation of tobacco products
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
2
Overview
1. Tobacco product regulation
2. Regional requirements and substances of interest
3. Tobacco product emissions
4. Regional trends for selected HPHCs in smoke
5. Evaluating method performance
6. Ruggedness and how to achieve it
7. Demonstrating continuing improvement
8. Opportunities for shared learning
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
Abbreviated list of HPHCs in
Cigarette Smoke
Acetaldehyde
Acrolein
Acrylonitrile
4-Aminobiphenyl
2-Aminonaphthalene
Ammonia
Benzene
Benzo[a]pyrene
1,3-Butadiene
Carbon monoxide
Crotonaldehyde
Formaldehyde
Isoprene
Nicotine
NNK
NNN
Toluene
Abbreviated list of HPHCs in
Smokeless Tobacco
Acetaldehyde
Arsenic
Benzo[a]pyrene
Cadmium
Crotonaldehyde
Formaldehyde
Nicotine
NNK
NNN
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
2. Classes of constituents – FDA TPSAC April 2012
Alkaloids Anabasine
Nicotine
Nornicotine
Alkenes /
cyanoalkanes 1,3-Butadiene
Isoprene
Hydrogen cyanide
Nitromethane
Aromatic amines 4-Aminobiphenyl
1-Aminonaphthalene
2-Aminonaphthalene
o-Anisidine
2,6-Dimethylaniline
o-Toluidine
Aromatics Benzene
Ethylbenzene
Toluene
Caffeic acid
Catechol
o-, m-, p-cresol
Phenol
Carbonyls Acetaldehyde
Acetone
Acrolein
Crotonaldehyde
Formaldehyde
Methyl ethyl ketone
Propionaldehyde
Heterocycles Quinoline
Styrene
N-nitrosamines 4-(Methylnitrosamino)-1-(3-pyridyl)-1-
butanone
N-Nitrosodiethanolamine (NDELA)
N-Nitrosodiethylamine
N-Nitrosodimethylamine (NDMA)
N-Nitrosomethylethylamine
N-Nitrosomorpholine (NMOR)
N-Nitrosonornicotine (NNN)
N-Nitrosopiperidine (NPIP)
N-Nitrosopyrrolidine (NPYR)
N-Nitrososarcosine (NSAR)
Heterocyclic aromatic amines A-α-C (2-Amino-9H-pyrido[2,3-b]indole)
Glu-P-1 (2-Amino-6-methyldipyrido[1,2-
a:3',2'-d]imidazole)
Glu-P-2 (2-Aminodipyrido[1,2-a:3',2'-
d]imidazole) CA
IQ (2-Amino-3-methylimidazo[4,5-
f]quinoline)
MeA-α-C (2-Amino-3-methyl)-9H-
pyrido[2,3-b]indole)
PhIP (2-Amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine)
Trp-P-1 (3-Amino-1,4-dimethyl-5H-
pyrido[4,3-b]indole)
Trp-P-2 (1-Methyl-3-amino-5H-
pyrido[4,3-b]indole )
Low MW
volatiles Acetamide
Acrylamide
Acrylonitrile
Ammonia
Ethyl carbamate
(urethane)
Ethylene oxide
Furan
Hydrazine
Propylene oxide
Vinyl acetate
Vinyl chloride
Trace metals Arsenic
Beryllium
Cadmium
Chromium
Cobalt
Lead
Mercury
Nickel
Polonium-210
Selenium
Uranium-235
Uranium-238
Polycyclic Aromatics Benz[a]anthracene
Benz[j]aceanthrylene
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Benzo[b]furan
Benzo[a]pyrene
Benzo[c]phenanthrene
Chrysene
Cyclopenta[c,d]pyrene
Dibenz[a,h]anthracene
Dibenzo[a,e]pyrene
Dibenzo[a,h]pyrene
Dibenzo[a,i]pyrene
Dibenzo[a,l]pyrene
Indeno[1,2,3-cd]pyrene
5-Methylchrysene
Naphthalene
Others Aflatoxin B1
Coumarin
Nitrobenzene
2-Nitropropane
Carbon monoxide
Chlorinated dioxins /
furans
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
3. Constituents of emissions from novel tobacco products
Substances associated with alternative nicotine / tobacco product emissions
5
3R4F MS smoke (whole)
Disposable E-cigarette aerosol
Heated Tobacco aerosol Single puff from each product
analysed using thermal
desorption, capillary gas
chromatography, TOF MS
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
3. Emissions from Electronic Cigarettes
Electronic cigarette aerosols contain very low levels of most HPHCs
Substances other than HPHCs may be present in e-cigarette aerosol, depending upon the e-liquid and device.
But are they present at levels significant to health?
6
1,3-Dioxan-5-ol
1-Cyanovinyl acetate
4-Methylbenzophenone
Abieta-8,11,13-trien-18 -oic acid
(dehydroabietic acid)
Acetaldehyde
Acetic acid
Acetic anhydride
Acrolein
β-nicotyrine
Benzene
Benzo[a]pyrene
Bisphenol A
Butyraldehyde
cis-Aconitic anhydride
Citraconic anhydride
Citric acid
Decanedioic acid, bis (2-ethylhexyl) ester
Ethanol
Ethyl acetate
Formaldehyde
Glycerol
Glycidol
Glyoxal
Hexadecanoic acid
(palmitic acid)
Isoprene
Itaconic anhydride
Maleic anhydride
Malic acid
Menthane (p-menthane)
Menthol
Menthone
Methyl 2-benzoylbenzoate
Methyl Acetate
Methylglyoxal
Methyl Vinyl Ketone
Nicotine
Octadecanoic acid (stearic acid)
Phenol
Propanoic acid
Toluene
Red = HPHC reported in e-cigarette aerosol
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
3. 3R4F smoke vs E-cigarette aerosol
HCI, comparison of selected constituents, scaled by consumption (14 cigarettes vs 300 puffs)
7
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
3R4F
E-cig
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
3. Emissions from tobacco heating products
Heated tobacco emissions are different to cigarette smoke
Tobacco heating product emissions contain lower levels of HPHCs than cigarette smoke.
They also contain other substances (as does cigarette smoke).
8
1,3-Butadiene
1-aminonaphthalene
2-Aminonaphthalene
3-Aminobiphenyl
4-Aminobiphenyl
Acetaldehyde
Acetone
Acrolein
Acrylonitrile
Ammonia
Benzene
Benzo[a]pyrene
Butyraldehyde
Carbon monoxide
Catechol
Crotonaldehyde
Formaldehyde
Hydrogen Cyanide
Hydroquinone
Isoprene
Methyl ethyl ketone
NAB
NAT
Nicotine
Nitric Oxide
NNK
NNN
o-Cresol
m+p-Cresol
Phenol
Propionaldehyde
Pyridine
Quinoline
Resorcinol
Styrene
Toluene
Red = detected in heated tobacco aerosol
Grey = awaiting data
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
3. 3R4F smoke vs heated tobacco aerosol vs E-cigarette vapour
HCI comparison of selected constituents per cig / consumable / 10 puffs
9
0,0
50,0
100,0
150,0
200,0
250,0
300,0
350,0
400,0
3R4F
HnB
E-Cig
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. Regional trends in selected smoke constituents
How similar are distributions in different regions?
Product testing results can be presented and interpreted in different ways. Fitness for purpose of data is influenced by the inter-laboratory agreement of results, i.e. Reproducibility.
The following slides present ‘snapshot data’ for selected constituents of mainstream smoke from commercial cigarettes from different regional markets
Do different regions have similar ranges of concentration? Would common ‘global’ methods be applicable? What is the impact of reproducibility on the ability to test products against a defined limit?
10
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. N-nitrosonornicotine in mainstream smoke, HCI
11
0
100
200
300
400
500
600
NN
N (
ng/
cig)
Product Index
Market A
0
100
200
300
400
500
600
NN
N (
ng/
cig)
Product Index
Market B
0
100
200
300
400
500
600
NN
N (
ng/
cig)
Product Index
Market C
0
100
200
300
400
500
600
NN
N (
ng/
cig)
Product Index
Market D
Graphs show: Mean Min Max, of replicate measurements by product, plus median
Data provided by Alison Eldridge,
BAT Group Research & Development.
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. 1,3-Butadiene in mainstream smoke, HCI
12
Graphs show: Mean Min Max, of replicate measurements by product plus median
20
40
60
80
100
120
140
160
1,3
-Bu
tad
ien
e (
µg/
cig)
Product Index
Market A
20
40
60
80
100
120
140
160
1,3
-Bu
tad
ien
e (
µg/
cig)
Product Index
Market B
20
40
60
80
100
120
140
160
1,3
-Bu
tad
ien
e (µ
g/ci
g)
Product Index
Market C
20
40
60
80
100
120
140
160
1,3
-Bu
tad
ien
e (
µg/
cig)
Product Index
Market D
Data provided by Alison Eldridge,
BAT Group Research & Development.
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. Benzo[a]pyrene in mainstream smoke, HCI
13
Graphs show: Mean Min Max, of replicate measurements by product and median
0
10
20
30
40
50
B[a
]P (
ng/
cig)
Product Index
Market A
0
10
20
30
40
50
B[a
]P (
ng/
cig)
Product Index
Market B
0
10
20
30
40
50
B[a
]P (
ng/
cig)
Product Index
Market C
0
10
20
30
40
50
B[a
]P (
ng/
cig)
Product Index
Market D
Data provided by Alison Eldridge,
BAT Group Research & Development.
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
5. Evaluating method performance
Specifying method performance requirements
14
Market Toxicant Units N Mean StdDev Min P25 Median P75 Max
A 1,3-Butadiene µg/cig 925 106.9 14.2 63.3 97.4 107.3 116.4 147.3
A B[a]P ng/cig 925 18.0 4.6 8.2 14.8 17.7 20.7 43.2
A NNN ng/cig 925 68.0 56.8 0.0 31.2 51.2 79.4 376.5
B 1,3-Butadiene µg/cig 695 70.8 16.2 31.4 60.3 68.1 79.3 126.3
B B[a]P ng/cig 700 18.6 4.9 7.1 15.2 18.1 21.3 42.2
B NNN ng/cig 700 139.1 52.4 31.0 104.0 135.5 163.0 423.0
C 1,3-Butadiene µg/cig 840 97.1 15.9 50.0 88.4 99.1 108.0 140.9
C B[a]P ng/cig 840 20.2 5.4 10.4 16.2 19.4 23.7 38.5
C NNN ng/cig 840 142.5 69.5 12.6 101.5 132.8 173.0 477.7
D 1,3-Butadiene µg/cig 700 98.8 20.5 51.8 83.1 101.3 112.9 156.5
D B[a]P ng/cig 700 13.1 3.9 5.1 10.4 12.7 15.3 30.3
D NNN ng/cig 700 150.4 78.1 33.2 102.9 133.4 174.4 515.2
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
5. Method performance requirement – 1,3-butadiene
15
20
40
60
80
100
120
140
160
1,3
-Bu
tad
ien
e (
µg/
cig)
Product Index
Market B
Market B median
75th percentile
To exclude with 95% confidence all products above the 75th percentile would
require a method reproducibility SD (sR) ≤ 3.99µg/cig
Method reproducibility limit = (79.3 – 68.1) µg/cig;
[1.96√2.sR] = 11.2µg/cig; sR = 3.99µg/cig;
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
1,3-butadiene reproducibility
16
Applying the reproducibility limit of CRM 70, ± 30.27 µg/cig
20
40
60
80
100
120
140
160
1,3
-Bu
tad
ien
e (
µg/
cig)
Product Index
Market B 2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
5. Performance of current methods
For a 10mg delivery product under HCI conditions, sR = 10.78µg/cig
Method reproducibility does not support the desired level of discrimination.
CORESTA Recommended Method No 70 Determination of selected volatile organic compounds in mainstream cigarette
smoke by GC-MS, July 2014
Mean = 73.77µg/cig; R = 30.27µg/cig; sR = 10.78µg/cig
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
5. Method performance requirement – NNN
18
Market D median
75th percentile
To exclude with 95% confidence all products above the 75th percentile would
require sR ≤ 14.6 ng/cig.
0
100
200
300
400
500
600
NN
N (
ng/
cig)
Product Index
Market D
R = 41ng/cig; [1.96√2sR] = 41ng/cig;
sR = 14.60ng/cig
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
5. Performance of current methods
sR = 12.7ng/cig : Reproducibility supports the desired level of discrimination.
If a decision is based upon a ratio (e.g. analyte/nicotine), the reproducibility of
measurement of nicotine must also be considered.
CORESTA Recommended Method No 75 Determination of tobacco-specific nitrosamines in mainstream cigarette smoke by LC-MS/MS,
July 2014
Sample 2 Mean = 87.5ng/cig;
R = 35.9ng/cig; sR = 12.7ng/cig
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
6. Rugged test methods
What makes a method able to tolerate minor changes without impacting performance?
Measurement by ratio – use a suitable internal standard (IS)
If low signal/noise, increase the selectivity of measurement - improve chromatographic separation; - use a more selective detection technique (e.g. MS, MS/MS) - remove matrix artefacts (cleanup).
For mass selective detection, use stable isotope dilution. Consider the number of unique ions required to assure adequate confidence in chemical identity
Apply system suitability criteria for selectivity and signal/noise
Consider technical guidance and data acceptance criteria applied to similar methods in other regulated industries
20
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
7. Demonstrating continuing improvement
Interlaboratory studies can meet different needs
Method performance studies, such as those conducted by CORESTA, enumerate the precision of measurement. In the absence of Certified Reference Materials, it is difficult to assess accuracy of measurement
21
Proficiency studies conducted in accordance with ISO/IEC 17043 and ISO 13528 evaluate the performance of participating laboratories against pre-established criteria by means of interlaboratory comparison. These include quantitative schemes for the measurement of constituents in test items.
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
7. Proficiency studies Can facilitate improved agreement of results
22
For example, by applying a target standard deviation based on fitness for purpose and
by collating information on methods to share best practice.
2007-2008 Annual Asia Collaborative Study on ISO Tar & Nicotine
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
Target Reproducibility [ RSDR ] can be calculated from the Horwitz function. The Horwitz function is based upon observations (drawn from thousands of collaborative studies) of the relationship between analyte concentration and reproducibility.
7. Predicting achievable performance
The Horwitz function
23
Reproduced from AMC technical Brief 17,
Royal Society of Chemistry 2004.
PRSDR (%) = 2 C-0.1505
HorRat = RSDR/PRSDR
The Horwitz function allows us
to estimate how reproducible
analytical methods can be,
providing a reality check.
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
8. Opportunities for shared learning
Best technical practice, internal and external quality assurance
Proficiency studies
- represent full range of product variation;
- address substances of technical priority (e.g. those of primary concern);
- operate regular cycles or rounds and provide accessible reports;
- generate commercial quality control samples;
- collate information on methods used (e.g. preparation, instrumental technique);
- generate information for learning, e.g. in technical workshops;
- apply standards consistent with best practice (e.g. a target sdR value).
Reference Materials
- we require definitive analytical methods to derive ‘reference’ values;
- homogeneity, long-term stability, continuous availability;
- can generate certified values for additional constituents over time;
- could be non-tobacco (e.g. smoke condensate).
24
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
Thank you for your attention
Questions?
www.bat-science.com
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. Mainstream Cigarette Smoke Constituents
4 regional markets were sampled and representative products tested
• Market A
– Mixed Blend, low incidence of charcoal in filter, Sampled Q1 2012
• Market B
– Mixed Blend, High incidence of charcoal in filter, Sampled Q4 2012
• Market C
– Predominantly FCV, Sampled Q1 2013
• Market D
– Predominantly US-Blended, Sampled Q4 2013, largest market
26
Thanks to my colleague Alison Eldridge of BAT Group Research & Development.
The data and a more complete discussion of their implications will be published
during 2014 and beyond.
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. Summary Statistics (4 markets combined)
Statistics for all replicates.
Products tested under HCI conditions
27
Market Toxicant Units N Mean StdDev Min P25 Median P75 Max
A 1,3-Butadiene µg/cig 925 106.9 14.2 63.3 97.4 107.3 116.4 147.3
A B[a]P ng/cig 925 18.0 4.6 8.2 14.8 17.7 20.7 43.2
A NNN ng/cig 925 68.0 56.8 0.0 31.2 51.2 79.4 376.5
B 1,3-Butadiene µg/cig 695 70.8 16.2 31.4 60.3 68.1 79.3 126.3
B B[a]P ng/cig 700 18.6 4.9 7.1 15.2 18.1 21.3 42.2
B NNN ng/cig 700 139.1 52.4 31.0 104.0 135.5 163.0 423.0
C 1,3-Butadiene µg/cig 840 97.1 15.9 50.0 88.4 99.1 108.0 140.9
C B[a]P ng/cig 840 20.2 5.4 10.4 16.2 19.4 23.7 38.5
C NNN ng/cig 840 142.5 69.5 12.6 101.5 132.8 173.0 477.7
D 1,3-Butadiene µg/cig 700 98.8 20.5 51.8 83.1 101.3 112.9 156.5
D B[a]P ng/cig 700 13.1 3.9 5.1 10.4 12.7 15.3 30.3
D NNN ng/cig 700 150.4 78.1 33.2 102.9 133.4 174.4 515.2
Total number of Products tested = 3165, replicates of 5
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. Benzo[a]pyrene in mainstream smoke, HCI, Market C
28
To distinguish between median and 75th percentile at 95% confidence Target Reproducibility Limit = 4.3 ng/cig
0
10
20
30
40
50B
[a]P
(n
g/ci
g)
Product Index
From CORESTA Recommended Method 58 (2014)
Reproducibility = 4.85 ng/cig (Sample 2, HCI, 18.37ng/cig mean)
Method reproducibility does not support discrimination
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
Benzo[a]pyrene method performance
29
0
10
20
30
40
50B
[a]P
(n
g/ci
g)
Product Index
Applying the CRM 58 Reproducibility Limit of ± 4.85 ng/cig
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
1,3-butadiene method performance
30
Applying the reproducibility limit of CRM 70, ± 30.27 µg/cig
20
40
60
80
100
120
140
160
1,3
-Bu
tad
ien
e (
µg/
cig)
Product Index
Market B 2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
NNN distribution and effect of method reproducibility
31
Median 133ng/cig; R upper 169ng/cig, R lower 97ng/cig
0
100
200
300
400
500
600N
NN
(n
g/c
ig)
Product Index
Market D 2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. Method stability 1,3-Butadiene
32
MAY11APR11MAR11FEB11JAN11DEC10NOV10OCT10SEP10AUG10
160
140
120
100
80
60
40
20
Month
1,3
-Bu
tad
ien
e (
µg
/cig
)Laboratory Control Cigarette, 3R4F
2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed
4. Method stability Benzo[a]pyrene
33
MAY11APR11MAR11FEB11JAN11DEC10NOV10OCT10SEP10AUG10
50
40
30
20
10
0
Month
B[a
]P (
ng
/cig
)
Laboratory Control Cigarette, 3R4F 2014
_TS
RC
95_W
right
TS
RC
2014
(68)
- D
ocum
ent n
ot p
eer-
revi
ewed