DETERMINATION OF FIFTEEN PRIMARY AND HETEROCYCLIC AROMATIC AMINES IN
MAINSTREAM CIGARETTE SMOKE USING LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY
CORESTA Congress, Chateau Frontenac, Quebec City, Canada, October 12 – 16, 2014
Spokes M. J.
Essentra Scientific Services, Shaftsbury Avenue, Jarrow, Tyne and Wear NE32 3UP, United
Kingdom
Summary
The levels of potentially harmful compounds in mainstream cigarette, smoke such as aromatic
amines, have been of interest for many years. GC-MS has previously been the most popular
method for determination of PAAs in cigarette smoke; however, the extraction and derivatisation
procedure is complex and time-consuming. LC-MS/MS, with higher selectivity and sensitivity,
permits a simplified clean-up procedure to be used. With increasing legislation, more aromatic
amines may be added to existing regulations and a simpler LC-MS/MS procedure has more
flexibility to accommodate this requirement.
Four aromatic amines 1AN, 2AN, 3AB, 4AB are required for Brazilian ANVISA and Health
Canada regulations, and o-anisidine, o-toluidine, 2,6-dimethylaniline and eight heterocyclic
aromatic amines (A-α-C, IQ, MeA-α-C, Trp-P-2, Trp-P-1, PhIP, Glu-P-1, Glu-P-2) have been
cited by the United States Food and Drug Administration (FDA) as harmful and potentially
harmful constituents (HPHC) in tobacco smoke. A single smoking and extraction method,
followed by LC-MS/MS methods for the analysis of all fifteen aromatic amines, is described.
Some HAAs have not been detected in reference cigarettes, so transfer rates were evaluated by
spiking components into the tobacco rod and collecting the smoke condensate on a Cambridge
filter (CF) pad. The procedure has also been shown to be applicable to e-cigarette vapour, and
data for e-cigarette products will be discussed.
Aromatic amines studied and abbreviations:
Hoffman Aromatic Amines (AA)
1-Aminonaphthalene
1AN
2-Aminonaphthalene
2AN
3-Aminobiphenyl
3AB
4-Aminobiphenyl
4AB
HPHC Aromatic Amines (ATD)
2,6-Dimethylaniline
2,6-D
o-Anisidine
o-Toluidine
o-A
o-T
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HPHC Heterocyclic Aromatic Amines
2-amino-α-carboline (2-amino-9H-pyrido[2,3b]indole
2-amino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole
2-aminodipyrido[1,2-a:3',2'-d]-imidazole
2-amino-3-methyl-3H-imidazo-[4,5-f]quinoline
2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine
2-amino-3-methyl-α-carboline (2-amino-3-methyl9H-pyrido[2,3-b]indole))
3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole
3-amino-1-methyl-5H-pyrido[4,3-b]indole
A-α-C
Glu-P-1
Glu-P-2
IQ
PhIP
MeA-αC
Trp-P-1
Trp-P-2
Standard analytical methods
The most widely used method follows the Health Canada GCMS method, with SPE and
derivatisation steps used for Hoffman aromatic amines.
Aims and objectives
A combined method for all the Hoffman and HPHC list aromatic amines was required, with
preference given to a single smoke and clean-up, as these are the most labour intensive stages.
Isotope labelled standards were required to give the best recovery and matrix effect
compensation.
Experimental
Materials and equipment
Cigarettes were smoked using a 20-port Linear smoking machine (SM450 Cerulean, Milton
Keynes, UK). Kentucky reference cigarettes (K3R4F) were provided by the Kentucky Tobacco
Research and Development Center (Lexington, KY, USA). SPE columns and a TurboVap®
Concentrator were supplied by Biotage (Cardiff, UK). Reference standards were provided by
Toronto Research Chemicals (Toronto, Ontario, Canada), Qmx Laboratories, (Essex, UK),
Sigma (St. Louis, MO, USA). LC-MS/MS equipment was an ABSciex API5000 (Framingham, MA
01701, USA). Separation was performed using a PrimesepD 150 x 3.2mm (SIELC) and a Luna
PFP(2) 150 x 4.6mm (Phenomenex).
Smoking and extraction procedure
Reference cigarettes were smoked under ISO 3308 and Health Canada Intense (HCI) smoking
conditions. The smoke condensate from 5 cigs (ISO) and 3 cigs (HCI) was trapped onto 44 mm
Cambridge filter (CF) pads. The CF pads were spiked with 100µl ISTD solution. The pads were
extracted into 20ml 0.1%HCl using an orbital shaker.
E-cigarettes were smoked with the regime, 55mL puff volume; 3sec puff duration; 2 puffs per
minute; square wave profile.
SPE clean-up for all samples was using 50mg SCX SPE (according to the manufacturers’
instructions), concentrated to 0.3mL.
This extract is then used for the analysis of Hoffman, HPHC and Heterocyclic aromatic amines
by three separate LC-MS/MS methods.
Spiked pad recoveries
K3R4F reference cigarettes were smoked under ISO and HCI conditions. Standard solutions
were spiked onto the pad prior to clean-up.
Transfer rates of Heterocyclic aromatic amines
To show that the trapping method employed is suitable to quantify a compound that is not
detected in the mainstream smoke, the transfer rates of four HAA (Glu-P-1, Glu-P-2, IQ and
PHiP) were evaluated by spiking K3R4F tobacco rods with known quantities of analyte. 25µl of a
125µg/mL standard solution was injected into the front of the tobacco rod to a depth of 20 – 30
mm; therefore, each cigarette contained nominally 3125ng of each amine. IQ is present in
smoke but was also spiked as a comparison.
As the experiment also allowed determination of the method uncertainty, filters were removed
from the cigarettes to increase levels of other analytes. ISO and intense regimes were smoked.
Transfer rates of aromatic amines from e-cigarettes
Twenty e-cigarettes with refillable cartomisers were filled with a typical e-liquid containing
propylene glycol, glycerol, nicotine and water. Concentrated standard solutions were added to
the liquid to give nominally; 12ng each Hoffman AA, 100ng each HPHC AA and 150ng each
HAA in 300mg e-liquid in each cartomiser.
Page 2 of 8
A 50 puff block was smoked and trapped on 44mm CFPs. The e-cigarettes were not smoked to
exhaustion.
LC-MS/MS
The three analysis methods are summarised below:
Agilent 1200
HPLC
Column
AA
SIELC
PrimesepD
150 x 3.2mm
Eluent A
Eluent B
Gradient
ATD
HAA
Luna PFP(2)
150 x 4.6mm
Luna PFP(2)
150 x 4.6mm
0.1% Acetic Acid (aq)
Acetonitrile
30%B
5.5mins 90%B
10%B
to 70%B 20mins
5%B
to 70%B
20mins
ABSciex API 5000 MS/MS
Ionisation mode
Positive ESI
The primesepD mixed mode column permits a relatively high acetonitrile mobile phase to be
used for Hoffman aromatic amines. This means interferents are eluted quickly and can be
diverted to waste. The Luna PFP gives good separation for anisidine, toliuidine and
dimathylaniline isomers. To separate the HAAs from their interferences requires a more aqueous
mobile phase, so a second run is used.
Results and Discussion
Hoffman aromatic amines
Aromatic amines and internal standards
LC-MS/MS cannot distinguish between isomers as they have the same base mass and
usually the same fragments. This means they have to be separated chromatographically by
retention time.
The three isomers of aminobiphenyl (green) elute in the order 3AB, 4AB, 2AB. 2aminobiphenyl is not required but has to be separated.
The isomers of aminonaphthalene (red) elute 2AN then 1AN
4AB-d3 (grey), 1AN-d3 and 2And3 (blue) are the internal standard chromatograms showing
similar retention times to the analytes.
Page 3 of 8
Aromatic amines and internal standards in a K3R4F
Analytes and internal standards clearly visible in the K3R4F trace. Interferents eluting
before 2.5 minutes are not seen as these have been diverted to waste.
K3R4F ISO Yields (ng/cig)
1AN
2AN
3AB
4AB
11.25
6.78
1.65
1.19
Previous Approaches
TSRC 2013 No55, Arista [1]
Beitrage Vol25, 2012 [2]
5.69
1.01
Essentra
Mainstream ISO
13.7
6.6
2.2
1.7
K3R4F HCI Yields (ng/cig)
1AN
2AN
3AB
4AB
Previous Approaches
Beitrage Vol25, 2012[2]
10.1
2.24
Essentra
Mainstream HCI
24.5
11
5
3.7
K3R4F Recovery (%)
1AN
2AN
3AB
4AB
Level 1
106
94.8
126.2
97.8
Level 2
112.7
90.3
104.1
109.3
HPHC aromatic amines
Anisidine, toluidine and dimethylaniline isomers
Page 4 of 8
Anisine isomers (green) elute in the order para, ortho, meta
Toluidine isomers (blue) elute in the order para, meta, ortho
2,6-Dimethlaniline (red) is the last peak to elute. The other isomers elute in a group before
11 minutes
Anisidine, toluidine and dimethylaniline analytes and internal standards
Anisidine (red, ISTD purple) , Toluidine(green, ISTD pink) and Dimethylaniline(dark blue,
ISTD light blue)
o-A, o-T, 2,6-D analytes and internal standards in K3R4F
Anisidine (grey, ISTD pale blue) , Toluidine(blue, ISTD red) and Dimethylaniline(green,
ISTD pink)
K3R4F ISO Yields (ng/cig)
2,6Dimethylaniline
o-Anisidine
o-Toluidine
3.2
2.34
46.38
2.32
54.1
4.0
2.6
47.9
2,6Dimethylaniline
o-Anisidine
o-Toluidine
4.25
99.3
5.7
94.8
Previous Approaches
TSRC 2013 No55 Arista [1]
Beitrage Vol25, No1, Feb 2012 [2]
Essentra
Mainstream ISO
K3R4F HCI Yields (ng/cig)
Beitrage Vol25, No1, Feb 2012 [2]
Essentra
Mainstream HCI
Page 5 of 8
8.5
2,6Dimethylaniline
o-Anisidine
o-Toluidine
Level 1 ISO
98
103
90
Level 2 ISO
100
104
88
Level 1 HCI
108
107
83
Level 2 HCI
109
109
80
K3R4F Recovery (%)
Heterocyclic aromatic amines
HAA analytes standard solution
----Glu-P-2 ----IQ ----Glu-P-1 ----Trp-P-2 ----PhIP ----Trp-P-1 ----A-α-C ----MeA-α-C
MeA-α-C (17mins) and Trp-P-2 (13mins) have the same transitions but are separated
chromatographically.
HAA internal standard solution
Elution order: IQ-d3, Trp-P-2 13C15N, PHiP-d3, AC-15N
Only four of the eight HAA have analogous internal standards. Price or availability prevented the use
of more.
Page 6 of 8
HAA analytes and ISTDs in a spiked K3R4F
----Glu-P-2 ----IQ ----Glu-P-1 ----Trp-P-2 ----PhIP ----Trp-P-1 ----A-α-C ----MeA-α-C
Analytes are grouped with their internal standards.
K3R4F ISO Yields
(ng/cig)
Ac
Glu-P 1
Glu-P-2
IQ
MeAC
PHiP
Trp-P-1
Trp-P-2
65.3
n.d.
n.d.
2.83
6.81
n.d.
1.49
2.55
77.8
n.d.
n.d.
2.2
6.8
n.d.
<LOQ
3.5
Ac
Glu-P 1
Glu-P-2
IQ
MeAC
PHiP
Trp-P-1
Trp-P-2
143
n.d.
n.d.
6.66
16.7
n.d.
3.61
6.38
210.7
n.d.
n.d.
48.3
20.5
n.d.
5.6
20.6
175.7
n.d.
n.d.
3.7
14.8
n.d.
<LOQ
6.8
Ac
Glu-P 1
Glu-P-2
IQ
MeAC
PHiP
Trp-P-1
Trp-P-2
Mainstream ISO
2.20%
2.20%
7.50%
2.50%
Mainstream HCI
4.60%
4.50%
14.10%
5.00%
Ac
Glu-P 1
Glu-P-2
IQ
MeAC
PHiP
Trp-P-1
Trp-P-2
Level 1 ISO
104.5
38.6
47.3
101
139.9
92.6
107.5
112.6
Level 2 ISO
92.6
40.9
46.6
104
128.5
90.8
108.1
105.7
Level 1 HCI
117.8
37.1
51
88.4
119.4
85.8
102.1
97.6
Level 2 HCI
113
38.1
47.9
91.6
125.1
90.2
107.9
99.3
Previous Approaches
Coresta 2012 [3]
Essentra
Mainstream ISO
K3R4F HCI Yields
(ng/cig)
Previous Approaches
Coresta 2012 [3]
TSRC 2013 No 15 [4]
Essentra
Mainstream HCI
Transfer Rates
K3R4F Recovery (%)
Spiked pad recoveries for Glu-P-1 and Glu-P-2 are low. These use IQ-d3 as an internal
standard. A recovery sample is run to correct for this, although these analytes are not normally
found.
E-cigarettes
Delivery from e-cigarettes is very variable. The e-TPM from 20 e-cigs varied from 80 to 210mg
for 50 puffs. Therefore the recovery is calculated as a percentage of the aromatic amine
expected for the delivery of TPM obtained. If the amines were transferring with the e-liquid
vapour we would expect 100% recovery.
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Average recovery
%
% Transferred (recovery) grouped by TPM
Amine Recovery Corrected for TPM
Delivery
Analyte
%
Analyte
%
1AN
75
A-C
61
2AN
89
Glu-P-1
60
3AB
75
Glu-P-2
50
4AB
72
IQ
49
Me-Ac
66
2,6Dimethylaniline
68
PHiP
3
o-Anisidine
71
Trp-P-1
57
o-Toluidine
68
Trp-P-2
39
50 puff block
e-TPM
80 to 210 mg
Recoveries (including Glu-P-1 and 2) are generally between 50 and 90% except for PHiP which
is not transferred at all. If the data is grouped by TPM into low (n=5, 112mg TPM), overall
average (n=20, 148mg TPM) and high (n=9, 189mg TPM), it can be seen that recovery
increases with higher TPM, suggesting a higher temperature is needed to vaporise the aromatic
amines.
Conclusion
LC-MS/MS is highly selective but can be plagued by invisible interference from matrix
suppression. A clean sample may not need much chromatographic separation or conversely, an
analyte in a dirty sample can be separated chromatographically from interference. In a
commercial lab the method must be optimised to provide the most efficient balance of these
approaches, and the mainstream method presented here achieves this.
References
1)
2)
3)
4)
5)
Martin A, Selected Aromatic Amines by Gas Chromatography Mass Spectrometry:
Challenges of Mainstream Cigarette Smoke, TSRC 2013, No55
Roemer, E. et al, Mainstream Smoke Chemistry abd In vitro and in Vivo Toxicity of
the Reference Cigarettes 3R4F and 2R4F Beitrage Vol 25, No1 February 2012
Bao, M.L., Wu, J.C., Rickert, W.S., Masters, A.P., Joza, P.J., Analysis of
Heterocyclic Aromatic Amines in Mainstream Cigarette Smoke Condensate by Solid
Phase Extraction and Ultra Performance Liquid Chromatography Tandem Mass
Spectrometry Coresta 2012 Congress, Paper SS-18
Desai,B.,Becker, U., Perumalla, N., Quantitative Analysis of Heterocyclic Aromatic
Amines in Mainstream Cigarette Smoke by Liquid Chromatography and Tandem
Mass Spectrometry TSRC 2013, No15
Volmer, D.A, Jessome, L.L. Ion Suppression a major concern in mass spectrometry;
LCGC North America Volume 24 Number 5 May 2006
Essentra's filter performance results shown here were obtained under controlled laboratory conditions, in accordance with ISO or
Essentra test methods (details available upon request), and are stated for Essentra's illustrative purposes only and should not be
relied upon by any other person for any reason. Essentra makes no representation or warranty as to the applicability of the test
results shown here or the suitability of the products described in this presentation to a customer's requirements.
Copyright 2014, Essentra Filters.
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