SUPPORTING INFORMATION
EXPERIMENTAL
Reagents and chemicals
Glyburide and glyburide-d11 internal standard were purchased from Sigma Aldrich (St. Louis,
MO, USA) and Toronto Research Chemicals (North York, Ontario, Canada), respectively.
Ultrapure LC/MS grade water and methanol were purchased from Avantor (Phillipsburg, NJ,
USA) and 99%-pure formic acid was obtained from EMD Millipore (Billerica, MA, USA).
Stock solutions were prepared at 1.0 mg/mL in ultrapure methanol. A 10X spiking solution
containing 10 µg/mL glyburide and 7.5 µg/mL glyburide and glyburide-d11 internal standard
was prepared in water. Rat plasma, lung, kidney, brain, eye, pancreas, small intestine and
stomach were purchased from Bioreclamation (Hicksville, NY, USA).
Sample preparation
Plasma extracts were prepared using rat plasma collected containing EDTA. Plasma proteins
were precipitated with an equal volume of ultrapure acetonitrile (ACN) followed by
centrifugation at 13k rpm in a tabletop centrifuge (Eppendorf, Hauppauge, NY, USA) for 30
min at 4°C. The supernatant fluid was decanted into a fresh microfuge tube and the contents
were evaporated to dryness under N2 gas using a TurboVap II concentration evaporator
workstation (Zymark AG, Allschwil, Switzerland). The resulting pellet was reconstituted in
the volume of deionized water containing 1000 ng/mL glyburide + 750 ng/mL internal
standard required to achieve the desired dilution of matrix components.
Rat tissue homogenates were prepared on ice in deionized water at 500 mg/mL using
a handheld Polytron™ homogenizer (Kinematica AG, Luzern, Switzerland). Dilutions of 1-
to 3000-fold of each tissue homogenate were prepared in deionized water spiked to yield final
concentrations of 1000 ng/mL glyburide and 750 ng/mL glyburide-d11 internal standard. A
standard blank (750 ng/mL internal standard alone) and a reference (1000 ng/mL glyburide +
750 ng/mL internal standard) prepared in deionized water were included as controls. Three by
one inch glass slides (VWR, Bridgeport, NJ, USA) were spotted with 2 µL of spiked
homogenate and were dried at room temperature in a glass vacuum desiccator (VWR,
Bridgeport, NJ, USA) prior to LESA-MS analysis.
Data analysis and interpretation
Mass spectrometric data capture and quantitation were done using Analyst version 1.5.2 and
MultiQuant version 2.0.2, respectively (Sciex, Concord, Ontario, Canada). Chemical
structures were rendered using Chemdraw Ultra 12.0 (Cambridge Soft, Waltham, MA, USA).
Tables were produced using Microsoft Excel 2010 (Redmond, WA, USA). Plots and statistics
were made with GraphPad Prism version 5.04 for Windows (San Diego, CA, USA).
Effect of glyburide spot volume on LESA-MS calibration curve linearity
The curve was generated by linear regression (n = 6) and weighting of 1/x. The lower limit of
quantitation (LLOQ) was 1 ng/mL and r2 values were 1.000, 0.9999, and 0.9999 for 2, 3 and
5 µL spots, respectively.
2 .5
3 L S pot
2 .0
( g ly b u r id e /IS )
P e a k A r e a R a t io
2 L S pot
5 L S pot
1 .5
1 .0
0 .5
0 .0
0
200
400
600
800
1000
N o m in a l C o n c e n t r a tio n
( n g /m L )
Ion-suppressive effects of extracted plasma on glyburide LESA-MS response
Suppression of glyburide ionization in reconstituted rat plasma extract is significant as
compared to control (neat solution). Glyburide response was restored to approximately 86%
of control by a 1500x dilution of plasma matrix components. One-way ANOVA with
Dunnett’s post-hoc test was performed. Differences considered significant if p <0.05 at the
95% CI level.
* * * P v a lu e < 0 .0 0 0 1 a t 9 5 % C I
100
% CTRL  SD
80
***
***
***
60
40
***
20
0
5
1
1
0
0
0
0
0
5
7
5
0
0
0
0
1
C
T
R
L
0
E x t r a c t e d p la s m a
( d ilu t io n f a c t o r )
Ion-suppressive effects of rat tissue homogenate dilutions on glyburide LESA-MS
response
One-way ANOVA with Dunnett's post-test was performed. Differences considered
significant if p <0.05 at the 95% CI level. The *, ** and *** labeling represents increased
95% CI of the difference.
S
D ilu t io n f a c t o r
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ta
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1 .5
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1 .0
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1 .5
S to m a c h
1 .0
***
***
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***
D ilu t io n f a c t o r
1 .5
Lung
1 .0
*
***
0 .0
***
D ilu t io n f a c t o r
1 .5
S m a ll In t e s t in e
1 .0
***
***
0 .0
***