The Analysis of Isotopically Labeled Propylene Glycol in ecigarettes

Transcription

1 The Analysis of Isotopically Labeled Propylene Glycol in ecigarettes Raymond H. Farmen, Ph.D.

2 The Coolest ecigarette Clinical Study Using Isotopically Labeled Propylene Glycol Raymond H. Farmen, Ph.D.

3 Presentation flow

4 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

5 Chemistry Stable Isotopes 5 Atom Identification # Protons # Neutrons AMU Hydrogen Hydrogen Deuterium* Tritium** Carbon Carbon C13* C14** > 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

6 Chemistry EXAMPLE - Stable Label Drug H H H D H H C C OH H D C C OH Slide 6 H H Ethanol Mass = 46 d 0 -ethanol H D H Ethanol Mass = 49 d 3 -ethanol Physical chemical properties d 0 -ethanol = d 3 -ethanol The only instrument/test that can distinguish between d 0 -ethanol and d 3 -ethanol is a mass spectrometer

7 Chemistry Stable Isotopes of Propylene Glycol (PG) H H H H C C C H OH OH H C 3 H 8 O 2 Atomic mass = 76 D D H D 13 C 13 C 13 C OH OH H H 13 C 3 H 5 D 3 O 2 Atomic mass = 82 Analytical Internal Standard H H H H 13 C 13 C 13 C OH OH H H 13 C 3 H 8 O 2 Atomic mass = 79 Carbon Heavy PG

8 Chemistry Mass Spectometer in Action Slide 8 = (m - 2) / z + = m/z that we want = (m + 2) / z + +

9 Method Summary Measuring PG in Human Plasma

10 PG LLOQ ng/ml in Ultrapure Water Sample Name: " ZZ STD B 1 1" Sample ID: "10" File: "ZZ RUN-052-V01.wiff" Peak Name: "Propylene Gl" Mass(es): " / Da" Comment: "none" Annotation: "" Sample Index: 10 Sample Type: Standard Concentration: 100. ng/ml 4600 Calculated Conc: 100. ng/ml Acq. Date: 8/18/ Acq. Time: 4:39:48 PM 4400 Modified: No Proc. Algorithm: Specify Parameters - MQ III 4300 Noise Percentage: 80 Base. Sub. Window: 1.00 min 4200 Peak-Split. Factor: 2 Report Largest Peak: No 4100 Min. Peak Height: cps Min. Peak Width: 0.00 sec 4000 Smoothing Width: 3 points RT Window: 30.0 sec 3900 Expected RT: 1.17 min Use Relative RT: No 3800 Int. Type: Base To Base 3700 Retention Time: 1.10 min Area: 8045 counts 3600 Height: 4512 cps 1.07 min Start Time: 3500 End Time: 1.17 min t R : 1.1 min Area: 8045 counts I n t e n s i t y, c p s Time, min

11 PG Calibration Curve: ,000 ng/ml ZZ PRO-V01-Presentation.rdb (Propylene Gl): "Linear" Regression ("1 / (x * x)" weighting): y = x (r = ) A n a ly te A re a / IS A re a e4 Analyte Conc. / IS Conc.

12 PG Concentration (ng/ml) PG Concentrations in Human Plasma n = 39 plasma lots Concentrations PG = ng/ml Plasma Lot

13 13 C 3 -PG Human Plasma Blank Sample Name: "9 020 ZZ Blank 1" Sample ID: "20" File: "ZZ RUN-009-V02.wiff" Peak Name: "13C3 Propylene Glycol" Mass(es): " / Da" Comment: "none" Annotation: "" Sample Index: 20 Sample Type: Unknown 2000 Concentration: N/A Calculated Conc: No Intercept 1950 Acq. Date: 8/22/2014 Acq. Time: 12:12:39 PM 1900 Modified: No Unlike PG there was no 13 C 3 -PG in human plasma! I n t e n s i t y, c p s Time, min

14 13 C 3 -PG LLOQ ng/ml in Plasma Sample Name: "9 010 ZZ STD B 1 1" Sample ID: "10" File: "ZZ RUN-009-V02.wiff" Peak Name: "13C3 Propylene Glycol" Mass(es): " / Da" Comment: "none" Annotation: "" Sample Index: Sample Type: Standard Concentration: 5.00 ng/ml Calculated Conc: 4.76 ng/ml 1950 Acq. Date: 8/22/2014 Acq. Time: 11:54:22 AM 1900 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: 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 1.23 min Retention Time: Area: 3387 counts Height: 1611 cps 1.20 min Start Time: End Time: 1.29 min I n t e n s i t y, c p s t R : 1.2 min Area: 3387 counts Observation: PG LLOQ = 100 ng/ml 13 C 3 PG LLOQ = 5 ng/ml This is a big peak at the LLOQ Time, min

15 13 C 3 -PG Calibration Curve ng/ml A n a ly te A re a / IS A re a ZZ C-009-V01-Presentation1.rdb (13C3 Propylene Glycol): "Linear" Regression ("1 / (x * x)" weighting): y = x e-015 (r = ) Analyte Conc. / IS Conc.

16 The power of stable isotopes in human studies APPLIED SCIENCE Not radioactive

17 Second Hand Exposure to ecigarettes ecigarette filled with 13 C 3 -PG solvent Only source of 13 C 3 -PG in the world If any 13 C 3 -PG shows up in this person s blood then it had to come from the ecigarette

18 Pilot ecigarette 13 C 3 -PG Study: - Study Product 98.2% carbon heavy PG

19 Pilot ecigarette 13 C 3 -PG Study: - Study Subjects All Subjects Healthy males years of age Vapers (n = 3) Non- Vapers (n = 3) 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 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

20 Pilot ecigarette 13 C 3 -PG Study: - Study Design

21 Estimated exposure by weight of vaping solution Subject C13C Propylene Glycol (98.2%) Nicotine (1.8%) mg 4.39 mg 2* 89.5 mg 1.64 mg mg 4.36 mg *Suspected device failure the subject experienced difficulty activating the device during some of the inhalations.

22 Nicotine Plasma Concentrations Nicotine C max from a single conventional cigarette = ng/ml

23 Unlabeled PG Plasma Concentrations MEAL Observation: All of these subjects had PG concentrations at the low end of the PG plasma continuum.

24 13 C 3 -PG Plasma Concentrations Vaping Subjects MEAL Observation: Dosed: 13 C 3 -PG / nicotine = 50 Cmax: 13 C 3 -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.

25 Second Hand Exposure to ecigarettes ecigarette filled with 98.2% 13 C 3 -PG Only source of 13 C 3 -PG in the world If any 13 C 3 -PG shows up in this person s blood then it had to come from the ecigarette How much 13 C 3 -PG showed up in this person s blood?

26 ( n g / m L ) 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: 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 Time, min 13 C 3 -PG Plasma Concentrations - Non-Vaping Subjects 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 No quantifiable concentrations were observed! LLOQ = 5 ng/ml Were any 13 C 3 -PG samples above the LOD? Sample Name: "9 010 ZZ STD B 1 1" Sample ID: "10" File: "ZZ RUN-009-V02.wiff" Peak Name: "13C3 Propylene Glycol" Mass(es): " / Da" Time (Minutes) I n t e n s i t y, c p s Subject C 3 -PG LLOQ Chromatogram LOD is 0.2 ng/ml

27 13 C 3 -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!

28 Conclusions