Measuring Hydrolytically Unstable Silanol for

Toxicokinetic Studies:

Overcoming Inherent Instabilities with New Methodology

Don Eldred and Chris Sushynski

ENC NMR Software Symposium: April 10th, 2016

From Polydimethylsiloxane (PDMS) to Silanol

• PDMS used in: shampoos, conditioners, skin care, polish

additives, waterproofing and many others

2

... Si O Si O Si O Si ...

OH2

OH Si O Si ...... Si O Si OH

• Biological contact with D4/D5 =

low molecular weight silanol

functional materials

• Soil contact with siloxanes =

low molecular weight silanol

functional materials

McMullin, T.S., Yang, Y., Campbell, J., Clewell, H.J., Plotzke, K., Andersen, M.E., Regul. Toxicol. Pharmacol., 74 (2016) S1-S13

Varaprath, S., McMahon, J.M., Plotzke, K.P., Drug Metab Dispos 31:206-204, 2003

Lehmann, R.G., Miller, J.R., Kozerski, G.E., Chemosphere 2000 Sep; 41 (5); 743-9

Pitfalls of Chromatographic Approach

• At concentrations in

recovered urine:

– Silanol condenses

during chromatographic

preparation

– Phenomenon is noted

in both GC (pictured)

and LC

3

Low MW Silanol

@ 2 ug/g

Low MW Silanol

@ 3600 ug/g

Condensed Silanol

Challenges of Low MW Silanol Analysis

1. When we dose Low MW Silanol at 15 wt % in a medium…

What is our confidence that it has not condensed?

2. When we retrieve samples, are we collecting metabolites

or equilibration products of metabolites?

3. When we prepare samples for measurements, are we

changing the equilibration conditions?

4. Do equilibration kinetics play a role during the course of

the measurement?

4

Why NMR of Low MW Silanol?

5

Does phase change drive

hydrolysis & condensation?

Does chromatography drive

hydrolysis & condensation?

No phase change.

No dilution.

No heating.

Measuring an Unadulterated Sample

• Target test article = recovered rodent urine

• 5 mm coaxial tube

• External tube = test article

• No concentration change

• No phase change

• No temperature change

6

Unmodified

Test Article

Aceto

ne-d

6

Toxicokinetic NMR: Establishing Confidence

• As a new analytical tool, we needed to establish confidence

– Concentration Linearity

– Limit of Quantification

– Molecular Selectivity

– Test Repeatability

– Repeatability

– Sample Stability when Frozen

– Hydrolysis and Condensation Kinetics

7

0.05 % 0.20 % 0.50 % 1.00 % 1.50 %

<1 h F,K,T,C,

Q,RK,T,C,R K,T,C,R K,T,C,R

F,K,T,C,

Q,R

F1, <1 h F F

F7, <1 h F F

F30, <1 h F F,C

11 h K,T,C,R K,T,C,R K,T,C,R K,T,C,R K,T,C,R

17 h K,T,C,R K,T,C,R K,T,C,R K,T,C,R K,T,C,R

27 h K,T,C,R K,T,C,R K,T,C,R K,T,C,R K,T,C,R

114 h K,T,C,R K,T,C,R K,T,C,R K,T,C,R K,T,C,R

127 h K,T,C,R K,T,C,R K,T,C,R K,T,C,R K,T,C,R

161 h K,T,C,RK,T,C,M

,RK,T,C,R K,T,C,R K,T,C,R

Profile of measures

R² = 0.998

0.00

0.25

0.50

0.75

1.00

1.25

1.50

0.00 0.25 0.50 0.75 1.00 1.25 1.50

Cal

cula

ted

Wt

% S

iloxa

nes

Predicted Wt % Siloxanes

Concentration Linearity: Total SiMe

• Range

0.05 to 1.5 wt %

Dosed into rodent urine

• Samples

5 concentrations

7 time points

• r2 = 0.998

• Relative Error = 5 to 8 %

8

Chemical Shift (ppm)0.010 0.009 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0 -0.001 -0.002 -0.003 -0.004 -0.005 -0.006 -0.007 -0.008

Chemical Shift (ppm)0.010 0.009 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0 -0.001 -0.002 -0.003 -0.004 -0.005 -0.006 -0.007 -0.008

Molecular Selectivity: Isolating principle silanols

• In water – near baseline resolution

• In Urine –

Two major silanol species converge

All signals broaden

Shim solutions vary in quality

9

In Water

In Urine

Limit of Quantification

• Commonly 10x RMS noise approximates LoQ

• Bad shims & high convolution limit this approximation

• Further complicated by kinetic evolution

– 1.5 wt % silanol measured 7 times @ 18-19 h

– 1.5 wt % silanol measured 3 times @ 0.5-1 h

– 0.05 wt % silanol measured 7 times @ 0.5, 11, 17, 27, 114, 127, & 161 h

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Concentration Relative error Method

>400 ppm ± 5-8* % Measured on 1.5 wt % silanol, low MW signal

55 ppm ± 30 % Measured on 0.05 wt % silanol, condensed silanol signal

1 ppm Unknown S/N extrapolation from 1.5 & 0.05 wt % prep samples

* - the designed study measures 1 %, but takes advantage of an unreasonable shim solution

Peak Detection: Deconvolution Solution

• Assume similar peak shapes

• If deconvolution is good, peak heights define concentration

• Deconvolution package

– 8 fitted points

– 0.25 Hz of peak position tolerance

– 0.04 to 2.5 Hz of peak width tolerance

– Non-Lorentzian fraction turned off

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Low MW Silanol Siloxane

Condensate

2J(1H,29Si)

Condensate2J(1H,29Si)

Low MW Silanol Siloxane

Inhomogeneity

Adjustment

Molecular Selectivity: Authentic vs Artifact

• Complexity of 1H SiMe region

– Various Me2Si-OH

– Spinning side bands

– 13C satellites

– 29Si satellites

– Other SiMe species

12

RU_1_5_gHMBCAD_01.fid.esp

F2 Frequency (Hz) 5 0 -5 -10 -15 -20 -25 -30 -35 -40 -45 -50

-24

-22

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

512x Z-axis expansion

Incr

easi

ng

sila

no

l MW

2 ppm F1 Sila

no

l fu

nct

ion

al P

DM

SB

ulk

PD

MS

1.35 Hz

Chemical Shift (ppm)0.15 0.10 0.05 0 -0.05 -0.10

Test Repeatability: Total SiMe

• 5 samples (0.05 to 1.5 wt %)

• 7 time measurements (0.2 to 165 h)

• Relative error ranges from 5 to 8 % per subset

• Water may be evaporating over a week yielding SiMe increase

• SiMe phase separation not detected

13

0.045

0.046

0.047

0.048

0.049

0.050

0.051

0.052

0.053

0.054

Wt

% S

iloxa

ne

Sample Age (hr)

0.5 11 17 27 110 130 160

±7.5

%

0.92

0.94

0.96

0.98

1

1.02

1.04

1.06

1.08

0 7 14 21 28 35

Wt

% S

ilan

ol S

iloxa

ne

by

1 H N

MR

wt

% S

ilan

ol S

iloxa

ne

Load

ed

Measurement Index

0.05 wt % 0.2 wt % 0.5 wt % 1.0 wt % 1.5 wt %

Low MW Silanol Stability @ -70 ⁰ C

• Prepared at 1.492 % low MW silanol

• Initial; +1 day frozen; +7 days frozen; +30 days frozen

• Acceptable freeze/thaw stability

• Acceptable 30 day storage

• Relative purity of low MW silanol >90 %

Measured at 1.55 ± 0.09 % silicone Low MW silanol relative to condensate = 96.5 ± 2.9 %

+1 DayInitial +7 Days +30 Days +1 DayInitial +7 Days +30 Days

Equilibration Kinetics

• Reaction follows simple:

• Significant change in X50 between 0.2 & 0.5 wt %

• Silanol condensation % is negligible @ 0.05 wt % dose

• Condensation % @ T∞ increases (up to 34 %) with silanol loading

15

kf

kr

A B C D+ +

1H NMR Capability Summary

• Linear SiMe detection between 0.05 & 1.5 wt %

• Repeatability = 5 to 8 % relative

• Limit of Quantification = 2 to 50 ppm, resolution limited

• Limit of Detection = 2 to 14 ppm, sensitivity limited

• Cryogenic stabilization demonstrated

• Low MW condensation equilibration kinetics characterized

• Synthetic standards & control spiking experiments not required

16

Acknowledgements

Jean Domoradzki, Ph.D., D.A.B.T.

Jacob Sushynski, B.S.

Greg Cushing, Ph.D.

Deb McNett, B.S.

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