analysis of n-nitrosodimethylamine (ndma) in … · 2.ndma-d6 as istd (0.5 ml, 200 ng/ml) 3.5 ml...
TRANSCRIPT
ANALYSIS OF N-NITROSODIMETHYLAMINE (NDMA) IN SMOKELESS TOBACCO BY
UHPLC-MS/MS
1
David Ericsson, Johan Lindholm
Swedish Match CORESTA, Berlin
2016-10-10
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
2 October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
5 October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
NDMA CHROMATOGRAM- PEAK SHAPE & SELECTIVITY
8
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
STANDARD 0.1 NG/ML
10
r.t. = 2.5 min
Vault switch
Zoom of chromatogram above
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
STANDARD CURVE
11
Range: 0.1-10 ng/ml Accuracy : 100-103 %
R2 = 0.999
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
ROUTINE USE OF THE METHOD
Over 10000 samples analyzed over the last 7 years
15
RSD of Control sample (Moist snuff A) = 10,3%
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
October 10, 2016
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A
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
2016
_ST
08_E
ricss
on.p
dfC
ongr
ess2
016
- D
ocum
ent n
ot p
eer-
revi
ewed
by
CO
RE
ST
A