The Analysis of Isotopically Labeled

Propylene Glycol in eCigarettes

Raymond H. Farmen, Ph.D.

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The Coolest eCigarette Clinical Study Using

Isotopically Labeled Propylene Glycol

Raymond H. Farmen, Ph.D.

2014

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Presentation flow

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Celerion’s Patent for Testing e-Cigarettes

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

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5

Chemistry 101

- Stable Isotopes

> 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

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

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Slide 6

Chemistry 101

- EXAMPLE - Stable Label Drug

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

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Chemistry 101

- Stable Isotopes of Propylene Glycol (PG)

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

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Slide 8

Chemistry 101

- Mass Spectometer in Action

+

+

+

= (m - 2) / z

= m/z that we want

= (m + 2) / z +

+

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Method Summary

Measuring PG in Human Plasma

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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 min

Peak-Split. Factor: 2

Report Largest Peak: No

Min. Peak Height: 300.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 3 points

RT Window: 30.0 sec

Expected RT: 1.17 min

Use Relative RT: No

Int. Type: Base To Base

Retention Time: 1.10 min

Area: 8045 counts

Height: 4512 cps

Start Time: 1.07 min

End Time: 1.17 min

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tR: 1.1 min Area: 8045 counts

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PG Calibration Curve: 100 – 10,000 ng/mL

ZZ41511-01-PRO-V01-Presentation.rdb (Propylene Gl): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000645 x + 0.00757 (r = 0.9997)

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PG Concentrations in Human Plasma

n = 39 plasma lots

10

100

1000

10000

0 5 10 15 20 25 30 35 40

PG

Co

nce

ntr

atio

n (

ng

/ml)

Plasma Lot

Concentrations PG = 40 – 4000 ng/ml

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13C3-PG Human Plasma Blank

Sample Name: "9 020 ZZ43723-01 Blank 1" Sample ID: "20" File: "ZZ43723-01-RUN-009-V02.wiff"Peak Name: "13C3 Propylene Glycol" Mass(es): "288.200/166.200 Da"Comment: "none" Annotation: ""

Sample Index: 20

Sample Type: Unknown

Concentration: N/A

Calculated Conc: No Intercept

Acq. Date: 8/22/2014

Acq. Time: 12:12:39 PM

Modified: No

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Unlike PG there was no 13C3-PG in human plasma!

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13C3-PG LLOQ - 5.00 ng/mL in Plasma

Sample Name: "9 010 ZZ43723-01 STD B 1 1" Sample ID: "10" File: "ZZ43723-01-RUN-009-V02.wiff"Peak Name: "13C3 Propylene Glycol" Mass(es): "288.200/166.200 Da"Comment: "none" Annotation: ""

Sample Index: 10

Sample Type: Standard

Concentration: 5.00 ng/mL

Calculated Conc: 4.76 ng/mL

Acq. Date: 8/22/2014

Acq. Time: 11:54:22 AM

Modified: No

Proc. Algorithm: Specify Parameters - MQ III

Noise Percentage: 50

Base. Sub. Window: 1.00 min

Peak-Split. Factor: 2

Report Largest Peak: No

Min. Peak Height: 100.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 3 points

RT Window: 30.0 sec

Expected RT: 1.22 min

Use Relative RT: No

Int. Type: Base To Base

Retention Time: 1.23 min

Area: 3387 counts

Height: 1611 cps

Start Time: 1.20 min

End Time: 1.29 min

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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

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13C3-PG Calibration Curve 5 – 1000 ng/mL

ZZ43723-01-13C-009-V01-Presentation1.rdb (13C3 Propylene Glycol): "Linear" Regression ("1 / (x * x)" weighting): y = 0.0125 x + -2.08e-015 (r = 0.9993)

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The power of stable isotopes in human studies

APPLIED SCIENCE

Not radioactive

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Second Hand Exposure to eCigarettes

• 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

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Pilot eCigarette 13C3-PG Study:

- Study Product

•

•

98.2% carbon heavy PG

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Pilot eCigarette 13C3-PG Study:

- Study Subjects

• Healthy males

• 26 – 37 years of age

All Subjects

• 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)

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Pilot eCigarette 13C3-PG Study:

- Study Design

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Estimated exposure by weight of vaping solution

Subject C13C 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

*Suspected device failure – the subject experienced difficulty activating the device during some of the inhalations.

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Nicotine Plasma Concentrations

Nicotine Cmax from a single conventional cigarette = 15 – 20 ng/ml

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Unlabeled PG Plasma Concentrations

MEAL

Observation: All of these subjects had PG concentrations at the low end of the PG plasma continuum.

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13C3-PG Plasma Concentrations

– Vaping Subjects

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.

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Second Hand Exposure to eCigarettes

• 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?

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13C3-PG Plasma Concentrations

- Non-Vaping Subjects

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

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7.0

LLOQ = 5 ng/ml

No quantifiable concentrations were observed!

Sample Name: "9 010 ZZ43723-01 STD B 1 1" Sample ID: "10" File: "ZZ43723-01-RUN-009-V02.wiff"Peak Name: "13C3 Propylene Glycol" Mass(es): "288.200/166.200 Da"Comment: "none" Annotation: ""

Sample Index: 10

Sample Type: Standard

Concentration: 5.00 ng/mL

Calculated Conc: 4.76 ng/mL

Acq. Date: 8/22/2014

Acq. Time: 11:54:22 AM

Modified: No

Proc. Algorithm: Specify Parameters - MQ III

Noise Percentage: 50

Base. Sub. Window: 1.00 min

Peak-Split. Factor: 2

Report Largest Peak: No

Min. Peak Height: 100.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 3 points

RT Window: 30.0 sec

Expected RT: 1.22 min

Use Relative RT: No

Int. Type: Base To Base

Retention Time: 1.23 min

Area: 3387 counts

Height: 1611 cps

Start Time: 1.20 min

End Time: 1.29 min

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13C3-PG LLOQ Chromatogram

LOD is 0.2 ng/ml Were any 13C3-PG

samples above the LOD?

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13C3-PG Extrapolated Plasma Concentrations below

LLOQ / above LOD – Non-Vaping Subjects

LLOQ = 5 ng/ml

Plasma Concentrations were 5000 X less than

that of Vapers!

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Conclusions

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