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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.
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Page 1: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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.

Page 2: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 3: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 4: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 5: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 6: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 7: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

Chemistry 101 - Mass Spectometer in Action

Slide 7

+

+

+

= (m - 2) / z

= m/z that we want = (m + 2) / z +

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Page 8: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 9: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

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Intensity, cps

9

tR: 1.1 min Area: 8045 counts

Page 10: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 11: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 12: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

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Intensit cps

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

Page 13: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

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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

Page 14: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

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12.0

12.5130

Page 15: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 16: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 17: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 18: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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)

Page 19: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 20: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 21: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

Page 22: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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.

Page 23: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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.

Page 24: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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?

Page 25: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

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Itit

13C3-PG LLOQ Chromatogram

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

the LOD?

Page 26: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

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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!

Page 27: The Analysis of Isotopically Labeled Propylene Glycol in ... · The Analysis of Isotopically Labeled Propylene Glycol in eCigarettes Raymond H. Farmen, Ph.D. Newland, K. E., Nachi,

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

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