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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
10
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
11
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.
17