Determination of Tobacco Specific

Haemoglobin Adducts in Smoking Mothers

and New Born Babies by Mass
Spectrometry
BY
Myers S.R. And Md. Yeakub Ali

Published by Biomarker Insight 2007:2,

269-282

Presented by - Under the guidance

Miss Aditi Patil of -
M.Sc. II Miss Swati Asani
 Introduction
• Content of tobacco smoke
– Most potent carcinogen
• NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]
• NNN [N-nitrosonornicotine]
• Nitrosation reaction
• Effect of tobacco smoke & nitrosamine
NNN NNK
Eosophageal carcinomas & Tumour of lung, liver,
Papillomas, carcinoma nasal cavity & pancreas
of nasal cavity & also leads to formation of
Hb-NNK adduct & DNA adduct
Objectives
• To determine Tobacco Specific Haemoglobin Adducts
of both African-American and Caucasian Smoking
Mothers and New Born Babies by Mass
Spectrometry.
• To study the effect of nitrosamine on smoking
mothers and newborn babies.
• To develop new sensitive method which would
involve simple extraction technique without a
chromatographic clean up prior to GC/MS operation.
Material
1] Sample from patients
2]Acetonitrile
3] Deuterated (3,3,4,4-D4) HPB
4] O-bis (trimethylsilyl)-trifluoroacetamide
[BSTFA]
5] 1N NaOH
6) 0.9% saline
7) Dichloroethane
8) N-hexane
9] 1N HCl
Methods
• Patient selection
– Labor and delivery patients
– 30% smokers
– From less than one pack per day to more than one
pack
• Sample preparation
• GC/MS analysis
Sample Preparation
10 ml of blood was collected in heparinized vacutainer during initial
admission of mother & from umbilical cord for measurement of
hemoglobin adduct

Stratification of samples according to smoking status

Blood samples are centrifuged at 3000 rpm for 10 min to generate packed
RBCs

RBCs were washed thrice with 0.9% saline & lysed by addition of 15 ml ice
cold deionized water with vigorous shaking
 Sample preparation for HPB-HB adduct
assay by GC/MS
100l lysed RBC was placed in 7 ml borosilicate glass tube

750 l HPLC grade water & 150 l 1N NaOH were added

10 l of 2.5 g/ml (3,3,4,4-D4)HPB was added as an internal standard

The solution was incubated in air at 50C for 2hrs

Hb solution was then acidified by addition of 225 l 1N HCl

Washed by addition of 2ml dichloroethane & 2ml N-hexane

Washed solution was neutralized by addition of 75 l of 1N
NaOH

Sample were extracted twice in 2 ml dichloroethane

Extracts combined & dried under a gentle stream

of N2 gas, & stored at -20C

Stock solution of HPB & deuterated (3,3,4,4-D4)HPB were prepared in

acetonitrile in conc. of 1.25, 5.0,12.5, 50.0, 125.0 & 500.0 ng/ml

Trimethylsilyl derivatization of HPB & deuterated HPB were prepared

by addition of BSTFA (10 l) & 30 l acetonitrile
 Incubation in air for 1 hr at 60 C

The derivatized samples were transferred to gas

chromatographic & mass spectrometric sample vials & were
diluted to a total volume of 100 l with acetonitrile

Analysis of samples

Positive Ion Negative Ion Electron Impact

Chemical Chemical Ionization
Ionization Ionization
GC/MS methods
HP 6890 was coupled to HP 5973 MS interfaced with Hewlett-packard
chemstation software

GC conditions were optimized with a temperature programming to attain the

highest sensitivity & resolution

Helium carrier gas was used with flow rate 1.3 ml/min

Injection of sample with split less injection mode with injection port
temperature at 280 C

Samples were eluted through GC column using temperature programme

consisting of initial temperature of 50C for 1 min followed by ramp
increase in temperature to 230 C.
 Results
Table 1: Quantification of HPB derivatized with BSTFA in maternal
and fetal cord blood samples using PICI mass spectrometry
Fig 1:Positive ion
chemical ionization
spectra of trimethylsilyl
derivatized 4-hydroxy-1-
(3-pyridyl)-1-butanone
[HPB] isolated from
maternal smokers blood
[(>1) pack/day]
[A]compared to the
spectra of authentic
derivatized HPB shown in
[B].
Table 2: Quantification of HPB derivatized with BSTFA in maternal
and fetal cord blood samples using NICI mass spectrometry
Fig 2: Negative ion
chemical ionization
spectra of trimethylsilyl
derivatized 4-hydroxy-1-
(3-pyridyl)-1-butanone
[HPB] isolated from
maternal smokers blood
[(> 1) pack/day] [A]
compared to the spectra
of authentic derivatized
HPB shown in [B].
Table 3: Quantification of HPB derivatized with BSTFA in
maternal and fetal cord blood samples using EI mass
spectrometry
Fig 3:Electron impact
ionization spectra of
trimethylsilyl
derivatized 4-hydroxy-
1-(3-pyridyl)-1-
butanone [HPB]
isolated from maternal
smokers blood [(>1)
pack/day] [A]compared
to the spectra of
authentic derivatized
HPB shown in [B].
Conclusion
• Similar values of both maternal & fetal nitrosamine adducts
were detected both African-American and Caucasian
populations
• Both chemical ionization techniques gave more accurate
results as compared to EI
• Placenta - Inefficient to block transfer of hazardous
chemicals
• Approximately 50% of maternal exposure to tobacco
specific nitrosamines was found to cross the placenta and
found adducted to fetal haemoglobin.
• This clearly indicate that fetal exposure to environmental
carcinogens can occur as a result of maternal exposures
during pregnancy.
Preventive measures
• Transformation of tobacco alkaloids by
Arthrobacter oxydans
• Inhibition of nitrosamine formation by ascorbic
acid
• Reduction in nitrosamine by Denitrifying bacteria
like Agrobacterium species
• Reduction in nitrosamine by organisms with no
nitrate reducing ability but having ability to
compete with nitrate reducing organisms
References
• Athrens, W., Brueske-Honfeld, I., Fortes C.,Boffetta,P., and Friesen, M.D. (1998) 4-
Hydroxy-1-(3-pyridyl)-1-butanone-hemoglobin adducts as biomarkers of exposure
to tobacco smoke: Validation of a method to be used in multicenter studies. Cancer
Epidemiol. Biomarkers Prev., 7(9):817-821.

• Ogawa,M., Oyama, T., Isse, T., Yamaguchi, T., Murakami, T., Endo, Y. and
Kayamoto, T. (2006) Hemoglobin adducts as a marker of exposure to chemical
substances, especially PRTR class I designated chemical substances. J. Occup.
Health., 48(5):314-328

• Schaffler G., Betz C., and Richter E.(1993) Mass spectrometric analysis of tobacco-
specific haemoglobin adducts. Environmental Health Perspectives, 99:187-189

• Shah, T., Sullivan, K. And Carter, J.(2006) Sudden infant death syndrome and
reported maternal smoking during pregnancy. Am. J. Public. Health., 96(10):1757-
1759

• Wagenknecht, L.E., Burke, G.L., Perkins, L.L., Haley, N.J. and Friedman G.D.
(1992) Misclassification of smoking status in the CARDIA study: a comparison of
self-report with serum cotinine levels. Am. J. Public. Health, 82(1):33-36
Thank you