The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes
Raymond H. Farmen, Ph.D. Newland, K. E., Nachi, R., Kafonek, C.J. and Islam, R.
Presentation flow
Celerion patent for testing eCigarettes Provisional patent filed September 9, 2013 Final patent filed September 9, 2014 (patent pending)
Chemistry 101 Stable isotopes LC-MS/MS
Describe a bioanalytical plasma assay for propylene glycol (PG) using LC-MS/MS
APPLIED SCIENCE The power of stable isotopes in clinical studies – 13C3-PG Design of an eCigarette pilot study using 13C3-PG
Results Conclusions
2
Celerion’s Patent for Testing e-Cigarettes
3
E-Cigarettes vaporize liquid nicotine in
solvent (propylene glycol or glycerol)
Problem: Solvents used are ubiquitous. Need to distinguish how much of this constituent get into the blood from the e-Cig, rather than the environment.
Patented Solution: Replace the solvent with a solvent that was made with stable isotopes (mass is greater)!! Use mass spectrometry to distinguish between endogenous levels of the constituents and those that came from the e-Cig
Chemistry 101 - Stable Isotopes
Atom Identification # Protons # Neutrons AMU
Hydrogen Hydrogen 1 0 1
Deuterium* 1 1 2
Tritium** 1 2 3
Carbon Carbon 6 6 12
C13* 6 7 13
C14** 6 8 14
4
> 99.99% of hydrogen on earth has an AMU = 1. > 99.9% of carbon on earth has an AMU = 12. An isotope of an atom has an atypical AMU. * stable isotope ** unstable isotope = radioactive
Chemistry 101 - EXAMPLE - Stable Label Drug
Slide 5
Ethanol Mass = 46
Ethanol Mass = 49
• Physical chemical properties d0-ethanol = d3-ethanol • The only instrument/test that can distinguish between d0-
ethanol and d3-ethanol is a mass spectrometer
C C
H
H
H
H
H
OH C C
H
H
H
H
H
OH
H
H
H
D
D
D
d0-ethanol d3-ethanol
Chemistry 101 - Stable Isotopes of Propylene Glycol (PG)
6
H C C C H
H H H
OH OH H
C3H8O2 Atomic mass = 76
H 13C 13C 13C H
H H H
OH OH H
13C3H8O2 Atomic mass = 79 Carbon Heavy PG
D 13C 13C 13C H
D D H
OH OH H
13C3H5D3O2 Atomic mass = 82
Analytical Internal Standard
Chemistry 101 - Mass Spectometer in Action
Slide 7
+
+
+
= (m - 2) / z
= m/z that we want = (m + 2) / z +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Method Summary Measuring PG in Human Plasma
Sample 0.05 mL plasma Add Analytical Internal Standard (13C3
2H3-PG)
Derivatization (to add some mass for LC-MS/MS) Liquid-liquid extraction of derivatives Reversed-phase gradient UHPLC separation Detection with AB Sciex API 4000™ tandem mass
spectrometer (ESI positive mode MRM) 8
PG LLOQ - 100 ng/mL in Ultrapure Water
Sample Name: "52 010 ZZ41511-01 STD B 1 1" Sample ID: "10" File: "ZZ41511-01-RUN-052-V01.wiff"Peak Name: "Propylene Gl" Mass(es): "285.100/163.200 Da"Comment: "none" Annotation: ""
Sample Index: 10 Sample Type: Standard Concentration: 100. ng/mL Calculated Conc: 100. ng/mL Acq. Date: 8/18/2014 Acq. Time: 4:39:48 PM Modified: No Proc. Algorithm: Specify Parameters - MQ III Noise Percentage: 80 Base. Sub. Window: 1.00 minPeak-Split. Factor: 2 Report Largest Peak: No Min. Peak Height: 300.00 cpsMin. Peak Width: 0.00 secSmoothing Width: 3 pointsRT Window: 30.0 secExpected RT: 1.17 minUse Relative RT: No Int. Type: Base To Base Retention Time: 1.10 minArea: 8045 countsHeight: 4512 cpsStart Time: 1.07 minEnd Time: 1.17 min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time, min
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
2600
2700
2800
2900
3000
3100
3200
3300
3400
3500
3600
3700
3800
3900
4000
4100
4200
4300
4400
4500
4600
Intensity, cps
9
tR: 1.1 min Area: 8045 counts
PG Calibration Curve: 100 – 10,000 ng/mL
10
0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4Analyte Conc. / IS Conc.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
64
PG Concentrations in Human Plasma n = 39 plasma lots
11
10
100
1000
10000
0 5 10 15 20 25 30 35 40
PG
Con
cent
ratio
n (n
g/m
l)
Plasma Lot
Concentrations PG = 40 – 4000 ng/ml
13C3-PG Human Plasma Blank
12
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
1800
1850
1900
1950
2000
Intensit cps
Unlike PG there was no 13C3-PG in human plasma!
13C3-PG LLOQ - 5.00 ng/mL in Plasma
13
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
1800
1850
1900
1950
2000
Itit
tR: 1.2 min Area: 3387 counts
Observation: • PG LLOQ = 100 ng/ml • 13C3–PG LLOQ = 5 ng/ml • This is a big peak at the LLOQ
13C3-PG Calibration Curve 5 – 1000 ng/mL
14
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000Analyte Conc. / IS Conc.
0.0
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
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
12.5130
The power of stable isotopes in human studies APPLIED SCIENCE
Not radioactive – therefore no licensing issues The stable isotope has the same physical/chemical
properties as the non-labeled compound. The mass of the stable label compound is usually
not endogenous Example - no 13C3-PG in blank human plasma
Study events that could affect the non-labeled compound will not affect the stable labeled compound.
15
Second Hand Exposure to eCigarettes
16
• eCigarette filled with 13C3-PG solvent
• Only source of 13C3-PG in the world
If any 13C3-PG shows up in this person’s blood then it had to come from the eCigarette
Pilot eCigarette 13C3-PG Study: - Study Product
KGo bottom-feeder tank with a single coil (~2.4 ohm resistance), 1100 mAh battery (~3.8 volts)
Approximately 1 mL of solution containing 1.8% nicotine and 98.2% carbon heavy PG loaded into the tank
17
Pilot eCigarette 13C3-PG Study: - Study Participants
18
• Healthy males • 26 – 37 years of age
All Participants
• 6-month product use history, daily product use for 30 days prior to the test visit
• No other nicotine products for the 14 days prior to the test visit
• 12 hour abstention prior to test visit product administration
Vapers (n = 3)
• No nicotine product use for 14 days prior to the test visit • Avoid environmental exposure to nicotine for 48 hours prior
to the test visit • USED TO TEST FOR SECOND HAND EXPOSURE
Non-Vapers (n = 3)
Pilot eCigarette 13C3-PG Study: - Study Design
Vaping participants self-administered one inhalation at least 3 seconds in duration every 30 seconds for a total of 30 inhalations.
All participants were confined to a small office (21.5 m3) for the first 2 hrs. of the study – confined space for testing second hand exposure
Collect blood samples for 8 hours from all participants and measure: nicotine PG 13C3-PG
19
Estimated exposure by weight of vaping solution
Participant 13C3 Propylene Glycol
(98.2%) Nicotine (1.8%)
1 239.4 mg 4.39 mg
2* 89.5 mg 1.64 mg
3 237.7 mg 4.36 mg
20
*Suspected device failure – the subject experienced difficulty activating the device during some of the inhalations.
Estimated exposure = (Pre-weight – Post-weight) X % compound
Nicotine Plasma Concentrations
Subject
1
2
3
4
5
6
Time (Minutes)
0 60 120 180 240 300 360 420 4
Nic
otin
e C
once
ntra
tion
(ng/
mL)
0
5
1
1
2
2
Nicotine Cmax from a single conventional cigarette = 15 – 20 ng/ml
Subject
1
2
3
4
5
6
Time (Minutes)
0 60 120 180 240 300 360 420 4
Pro
pyle
ne G
lyco
l Con
cent
ratio
n (n
g/m
L)
5
1
1
2
2
3
Unlabeled PG Plasma Concentrations
MEAL
Observation: All of these subjects had PG concentrations at the low end of the PG plasma continuum.
13C3-PG Plasma Concentrations – Vaping Participants
Subject
1
2
3
Time (Minutes)
0 60 120 180 240 300 360 420 4
13C
3 P
ropy
lene
Gly
col C
once
ntra
tion
(ng/
mL)
0
5
1
1
2
2
3
3
4
4
MEAL
Observation: Dosed: 13C3-PG / nicotine = 50 Cmax: 13C3-PG /nicotine > 150 The lung absorption of PG is 3X greater than that of nicotine
Half-life PG = 2 hrs. It would be impossible to calculate this without dosing a stable isotope.
Second Hand Exposure to eCigarettes
24
• eCigarette filled with 98.2% 13C3-PG
• Only source of 13C3-PG in the world
If any 13C3-PG shows up in this person’s blood then it had to come from the eCigarette
How much 13C3-PG showed up in this person’s blood?
13C3-PG Plasma Concentrations - Non-Vaping Participants
Subject 4 5 6
Time (Minutes) 0 60 120 180 240 300 360 420 480
1 3 C
3 P
r o p y
l e n e
G l y
c o l C
o n c e
n t r a
t i o n
( n
g / m
L )
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
LLOQ = 5 ng/ml
No quantifiable concentrations were observed!
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
1800
1850
1900
1950
2000
Itit
13C3-PG LLOQ Chromatogram
LOD is 0.2 ng/ml Were any 13C3-PG samples above
the LOD?
Subject
4
5
6
Time (Minutes)
0 60 120 180 240 300 360 420 4
13C
3 P
ropy
lene
Gly
col C
once
ntra
tion
(ng/
mL)
0
1
2
3
4
5
6
7
13C3-PG Extrapolated Plasma Concentrations below LLOQ / above LOD – Non-Vaping Participants
LLOQ = 5 ng/ml
Plasma Concentrations were 5000 X less than
that of Vapers!
Conclusions
We have developed and validated a LC-MS/MS assay for PG and Carbon Heavy PG
PG is ubiquitous in the environment PG plasma levels vary greatly between subjects PG plasma levels are affected by diet Carbon Heavy PG is a powerful tool for testing eCigarettes
The PG/nicotine absorption from the lungs of vapers is not linear. Carbon Heavy PG is a super powerful tool for testing second
hand exposure to eCigarettes Using the Carbon Heavy PG test could have a positive affect
on eCigarette legislation
27