1. Determination of Cotinine in Submadibular and Parotid Fluids by LC/MS/MS after Ingestion of NicotineDetermination of Cotinine in Submadibular and Parotid Fluids by LC/MS/MS after Ingestion of Nicotine.y g
R Sam Niedbala (san204@lehigh edu) and Jesús M GonzálezR. Sam Niedbala (san204@lehigh.edu) and Jesús M. González
Department of ChemistryDepartment of Chemistry
Lehigh UniversityLehigh University
1 Ab t t1. Abstract
LC/MS/MS method Samples analysisLC/MS/MS method Samples analysis
P i t d h d k i i t d ithPassive exposure to second hand smoke is associated withp
various illnesses ranging from allergies to cancers C ti i d ti i d3 t d d l ti F ll i i f d t thvarious illnesses ranging from allergies to cancers. Cotinine and cotinine d3 standard solutions Following informed consent, three non
Assessment of second hand smoke exposure, particularly
Cotinine and cotinine d3 standard solutions
d d d t t
Following informed consent, three non
ki l t h lf t d
Assessment of second hand smoke exposure, particularly
l bl l ti h hild ll were prepared and used to create a smoking volunteers who self-reported noamong vulnerable populations, such as children may allow were prepared and used to create a smoking volunteers who self reported nog y
intervention and prevention of future disease A rapid test to calibration curve MRM methods were exposure to SHS in the previous 48 hoursintervention and prevention of future disease. A rapid test to
d t i d h d k ld tit t
calibration curve. MRM methods were exposure to SHS in the previous 48 hours
determine second hand smoke exposure would constitute a
developed to monitor the 177 136/79 800 ingested 2 mg of an over the counter
p
valuable tool for researchers and clinicians Oral fluids are an developed to monitor the 177.136/79.800 ingested 2 mg of an over-the-countervaluable tool for researchers and clinicians. Oral fluids are an p
d 177 136/98 000 t iti f ti i
g g
i ti t bl t Wh l l fl id lexcellent sample matrix for this purpose, due to their versatility and 177.136/98.000 transitions for cotinine nicotine tablet. Whole oral fluid samplesexcellent sample matrix for this purpose, due to their versatility
d i i t h l fl id t d
and 177.136/98.000 transitions for cotinine nicotine tablet. Whole oral fluid samples
and non invasive nature; however, as oral fluids are secreted
and the 180 126/79 900 and were then collected by expectoration intoby different glands in the oral cavity a basic understanding of
and the 180.126/79.900 and were then collected by expectoration intoby different glands in the oral cavity, a basic understanding of
h t ll t th i i d i d t d i b tt 180 126/101 00 transitions for cotinine d3 vials at intervals over 5 hours Subhow to collect them is required in order to design better 180.126/101.00 transitions for cotinine d3 vials at intervals over 5 hours. Sub-q g
sample collectors and tests aimed at reducing the incidence of d i t l t d d El t dib l fl id ll t d isample collectors and tests aimed at reducing the incidence of used as internal standard. Electrospray mandibular fluids were collected using a
false positive results. Objectives: to determine the distribution
used as internal standard. Electrospray
d i i ti Th t i
mandibular fluids were collected using a
d i t il d f h i di id l tid
false positive results. Objectives: to determine the distribution
f ti i j t b lit f i ti th i was used as ionization source The curtain device tailored for each individual parotidof cotinine, a major metabolite of nicotine, among the various was used as ionization source. The curtain device tailored for each individual, parotidj g
glandular secretions that form the oral fluids Methods: gas (CUR) was set at 20 0 collision fluids were collected using a custom madeglandular secretions that form the oral fluids. Methods:
F ll i i f d t ki l t h lf
gas (CUR) was set at 20.0, collision fluids were collected using a custom-made
Following informed consent, non-smoking volunteers who self-
gas(CAD) was set at 5 Ionspray voltage device Pre dose samples were also
g , g
reported no exposure to second hand smoke in the previous gas(CAD) was set at 5, Ionspray voltage device. Pre-dose samples were alsoreported no exposure to second hand smoke in the previous g ( ) , p y g
(IS) t 5500 T t (TEM)
p
fi d t b ti f i ti48 hours, ingested 2 mg of over the counter nicotine. Using a (IS) at 5500, Temperature (TEM) was confirmed to be negative for nicotine48 hours, ingested 2 mg of over the counter nicotine. Using a
t t i di id l l d fl id l
(IS) at 5500, Temperature (TEM) was confirmed to be negative for nicotine
custom apparatus, individual gland fluid samples were
700 0 Ion source gas 1 (GS1) was 50 ion metabolites using LCMSMS and thereforeg
collected at intervals over 5 hours Pre-dose and post-dose
700.0, Ion source gas 1 (GS1) was 50, ion metabolites using LCMSMS and thereforecollected at intervals over 5 hours. Pre-dose and post-dose
l l t l d b LC/MS/MS d t i i th i source gas 2 (GS2) was 60 the entrance in agreement with volunteers self reportsamples were later analyzed by LC/MS/MS determining their source gas 2 (GS2) was 60, the entrance in agreement with volunteers self-report.p y y g
cotinine concentrations Pre dose samples were confirmed to
g ( ) ,
t ti l (EP) t t 11 th lli i
g p
C ll t d l th l dcotinine concentrations. Pre-dose samples were confirmed to potential (EP) was set at 11, the collision Collected samples were then analyzed
be negative and therefore in agreement with self-report.
potential (EP) was set at 11, the collision
it t ti l (CXP) t t 4 0 Th
Collected samples were then analyzed
i th LC/MS/MS th d d ib d
be negative and therefore in agreement with self report.
R lt Th ti i t ti k d ft th h exit potential (CXP) was set at 4 0 The using the LC/MS/MS method describedResults: The cotinine concentrations peaked after three hours; exit potential (CXP) was set at 4.0. The using the LC/MS/MS method described
however the maximum values varied from volunteer to compound dependent voltages are shown before The results for each volunteer arehowever, the maximum values varied from volunteer to
l t i f 16 t 36 / L N j diff
compound dependent voltages are shown before. The results for each volunteer are
volunteer ranging from 16 to 36 ng/mL. No major differences
in the table below shown in the figures below
g g g j
were observed among the different fluids analyzed suggesting in the table below. shown in the figures below.were observed among the different fluids analyzed, suggesting g
that cotinine is distributed uniformly among the differentthat cotinine is distributed uniformly among the different
ti th t f th l fl id C l i ti i i Volunteer 1 Cotinine distribution in oral fluidssecretions that form the oral fluids. Conclusions: cotinine is
Collision
Volunteer 1, Cotinine distribution in oral fluids
distributed uniformly among the different glandular fluids Dwell Declustering
Collision
Collision 40distributed uniformly among the different glandular fluids
t t d th f ll ti f l fl id f t ti
Q1 mass Q3 mass
Dwell
i
Declustering
i l
entrance
Collision
E
40
tested, therefore collection of oral fluids for testing can use
Q1 mass
(D )
Q3 mass
(D )
time potential
entrance
t ti l
Energy 36
l)
, g
either parotid or submandibular fluids
(Da) (Da)
(ms)
p
(DP)
potential
gy
(CE)
36
/m
either parotid or submandibular fluids . (ms) (DP)
(CEP)
(CE) 32
ng/
(CEP)
28
n(
2 I t d ti
28
tion
2. Introduction 177 136 79 800 150 0 46 00 12 00 31 00 24
trat
2. Introduction 177.136 79.800 150.0 46.00 12.00 31.00 24
ent
Parotid
20
nce
Submandibular
177 136 98 000 150 0 46 00 12 00 27 00
20
con
Wh l S li
Saliva is produced in and secreted from various
177.136 98.000 150.0 46.00 12.00 27.00 16
ec
Whole Saliva
Saliva is produced in and secreted from various
12
nin
glands People have three major pairs of salivary 180 126 79 900 150 00 51 00 10 00 35 00
12
otin
glands. People have three major pairs of salivary 180.126 79.900 150.00 51.00 10.00 35.00
8
Co
glands: the parotid submandibular and
8
glands: the parotid, submandibular, and 180 126 101 000 150 0 51 00 10 00 25 00 4
sublingual
180.126 101.000 150.0 51.00 10.00 25.00 4
sublingual. 0
0 1 2 3 4 5 6
An allure C18 column from Restek Time (hours)
An allure C18 column from Restek Time (hours)
(Bellefonte PA) was used in the HPLC(Bellefonte, PA) was used in the HPLC( , )
th d S l t A t t i i
Volunteer 2, Cotinine distribution in oral fluids
method. Solvent A was water containing 40method. Solvent A was water containing 40
0 1% formic acid and solvent B was 360.1% formic acid and solvent B was 36
)
methanol HPLC grade (Sigma) containing 32
/ml
methanol HPLC grade (Sigma) containing 32
ng/
g ( g ) g
0 1% f i id Th fl t 28
n(n
0.1% formic acid. The flow rate was 28
ion
0.1% formic acid. The flow rate was
i t i d t 0 2000 l/ i th l
24
rati
Parotid
maintained at 0 2000 ml/min the column
24
entr
maintained at 0.2000 ml/min, the column 20
nce
Submandibular
compartment was set at 30°C and the
20
con
Whole Saliva
compartment was set at 30 C, and the 16
ec
Whole Saliva
gradient program used was:
16
nine
gradient program used was: 12
otin
g p g 12
Co
8
C
Time (min) %B
8
Time (min) %B 4
Figure 1 - On average 75% of the fluid in the mouth 1 0 10
4
Figure 1 On average, 75% of the fluid in the mouth
f b dib l t i l
1.0 10 0
comes from submandibular mucous materials. 4 0 95
0
0 00 1 00 2 00 3 00 4 00 5 00 6 004.0 95 0.00 1.00 2.00 3.00 4.00 5.00 6.00
Th bj ti f thi k i t h l b ild
12.0 95 Time (hours)
The objective of this work is to help build a
12.0 95
13 0 2
( )
The objective of this work is to help build a 13.0 2
basic understanding of how to collect and 15 0 2
Volunteer 3, Cotinine distribution in oral fluids
basic understanding of how to collect and 15.0 2
test oral samples for detecting second 15 5 10
40
test oral samples for detecting second 15.5 10p g
h d k i t t d 17 0 10 36
)
hand smoke exposure using targeted 17.0 10
ml)
hand smoke exposure using targeted
l d ll ti Th l ti f th l d
32
ng/m
gland collection The location of the glands
n(n
gland collection. The location of the glands 28
ion
in the oral cavity is shown in Figure 1
rati
in the oral cavity is shown in Figure 1. 24
ntr
Parotid
Using a custom apparatus parotid and
ce
Submandibular
4 R lt
Using a custom apparatus, parotid and 20
on
Wh l S li
4. Results
g pp , p
b dib l fl id l
eco
Whole Saliva
4. Results
sub-mandibular fluid samples were 16
ine
sub mandibular fluid samples were 16
tin
collected from volunteers who had 12
Cot
LC/MS/MS calibration
collected from volunteers who had 12
C
LC/MS/MS calibration
previously ingested 2 mg of over the 8
Different solutions were prepared in the
previously ingested 2 mg of over the 8
Different solutions were prepared in the
p y g g
t i ti Th l 4p p
f 0 t 100 / L d d t
counter nicotine. These samples were 4
range from 0 to 100 ng/mL and used to
counter nicotine. These samples were
ll t d t l i t l d l d
0range from 0 to 100 ng/mL and used to
collected at regular intervals and analyzed
0
0 00 1 00 2 00 3 00 4 00 5 00 6 00
create a calibration curve for cotinine
collected at regular intervals and analyzed 0.00 1.00 2.00 3.00 4.00 5.00 6.00
create a calibration curve for cotinine,
via liquid chromatography mass Time (hours)
using cotinine d3 as internal standard
via liquid chromatography-mass Time (hours)
using cotinine d3 as internal standard.
spectrometry (LC/MS/MS) for cotinine a
g
R l ti ffi i t t th 0 999
spectrometry (LC/MS/MS) for cotinine, a
Relation coefficients greater than 0.999
p y ( ) ,
j t b lit f i ti
Relation coefficients greater than 0.999
bt i d Th lt h i
major metabolite of nicotine.
were obtained The results are shown in
major metabolite of nicotine.
were obtained. The results are shown in
the figure belowthe figure below
5 Conclusions5. Conclusions
3 Experimental method No significant difference was seen forLC/MS/MS calibration curve for cotinine in saliva3.Experimental method No significant difference was seen forLC/MS/MS calibration curve for cotinine in salivap
cotinine in submandibular or parotid fluids100 cotinine in submandibular or parotid fluids100
O l Fl id C ll ti
p
Oral Fluids Collection 90
Aft i i i f d t i C ti i ld th f b ll t d f
90
After signing informed consent, specimens were Cotinine could therefore be collected fore s g g o ed co se , spec e s e e
ll t d f 3 l t bj t b f d
Cotinine could therefore be collected for80
collected from 3 volunteer subjects before and analysis from anywhere in the mouthy = 105 36x 1 1487
y = 88.651x - 1.2235
R² = 0 9998collected from 3 volunteer subjects before and
ft th i t d 2 f Ni ti f
analysis from anywhere in the mouth.y = 105.36x - 1.1487
R² = 0.9997
R = 0.9998
70
)
after they ingested 2 mg of Nicotine from an
70
ml
after they ingested 2 mg of Nicotine from an
th t d t C it V l t
g/m
80 Da
over the counter product- Commit. Volunteer
C ti i l i b LCMSMS
60
ng
80 Da
Fragmentover the counter product Commit. Volunteer
k d f i f i d i ki
Figure 2 Cotinine calibration curve Cotinine analysis by LCMSMS was a
n(
Fragment
were asked to refrain from eating or drinking
Figure 2 – Cotinine calibration curve Cotinine analysis by LCMSMS was a
50
on
98 Dawere asked to refrain from eating or drinking
reliable technique for quantitative
50
atio
98 Da
Fragment
anything for 10 minutes prior to collections reliable technique for quantitative
tra
Fragment
anything for 10 minutes prior to collections.
determinations40
nt
Samples were collected 30 minutes prior to determinations
ce
Samples were collected 30 minutes prior to
30
onc
ingestion and immediately before ingestion
30
co
ingestion and immediately before ingestion.
6 A k l d t
ec
Following ingestion specimens were collected 6. Acknowledgments20
ine
Following ingestion, specimens were collected 6. Acknowledgments
ni
g g p
over a period of 5 hours All specimens were 10
oti
over a period of 5 hours. All specimens were 10
Co
p p
stored on ice and analyzed within 24 hours of NYU College of Dentistry Professor Dan
C
stored on ice, and analyzed within 24 hours of NYU College of Dentistry, Professor Dan0, y
collection
g y,
M l d D Willi Ab Ch l0 0 2 0 4 0 6 0 8 1 1 2collection. Malamud, Dr William Abrams, Cheryl0 0.2 0.4 0.6 0.8 1 1.2
Ratio (Sample/Internal standard) Malamud, Dr William Abrams, Cheryl
B b
Ratio (Sample/Internal standard)
BarberBarber