SUPPORTING INFORMATION
Automated high-capacity on-line extraction and bioanalysis of dried blood
spot samples using liquid chromatography/high-resolution accurate mass
spectrometry
Regina V. Oliveira1†, Jack Henion1* and Enaksha R. Wickremsinhe2
1
Quintiles Bioanalytical and ADME Laboratories, 19 Brown Rd., Ithaca, NY 14850, USA
Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN
2
46285, USA
*Correspondence to: J. Henion, Quintiles Bioanalytical and ADME Laboratories, 19 Brown Rd.,
Ithaca, NY 14850, USA.
E-mail: henionj@advion.com
†Permanent address: Chemistry Department, Federal University of São Carlos, São Carlos, SP,
13565-905, Brazil.
S-1
Figure S1. Relative MS response (analyte/SIL-IS) obtained from different mixtures of solvents tested for the on-line DBS extraction (n = 4) and respective relative
standard deviation (RSD).
S-2
Midazolam
15 ng/mL
5 ng/mL
18000
16000
MS Response
14000
12000
10000
8000
6000
4000
2000
0
1
2
3
Sequence of Extraction
-Hydroxymidazolam
15 ng/mL
4
5
4
5
5 ng/mL
4500
4000
MS Response
3500
3000
2500
2000
1500
1000
500
0
1
2
3
Sequence of Extraction
Figure S2. Representative MS response (n = 2; peak area/counts) showing five successive on-line
extractions and bioanalysis taken from the same DBS sampling area. Human DBS samples of midazolam
and α-hydroxymidazolam (15 and 5 ng/mL); extraction time of 24 s, using an extraction mixture
consisting of methanol/acetonitrile (50:50 v/v) containing 0.1% ammonium hydroxide.
S-3
Table S1. MS response (peak area/counts) of successive extractions taken from the same DBS sampling
area using the on-line DBS extraction and LC/HRMS analysis
Midazolam
Extraction
no.
Average
MS
response
(n =2)
1
2
3
4
5
5486
2460
2074
1563
428
5 ng/mL
RSD (%)
12.1
7.4
4.3
4.7
20.2
Individual
MS
response
compared
to total MS
response
(%)
45.6
20.5
17.3
13.0
3.6
Average
MS
response
(n =2)
16685
9967
5136
2123
1831
15 ng/mL
RSD (%)
10.3
4.0
9.7
0.2
3.4
Individual
MS
response
compared
to total MS
response
(%)
46.7
27.9
14.4
5.9
5.1
Table S2. MS response (peak area/counts) of successive extractions taken from the same DBS sampling
area using the on-line DBS extraction and LC/HRMS analysis
α-Hydroxymidazolam
Extraction
no.
Average
MS
response
(n =2)
1
2
3
4
5
1593
1029
318
228
142
5 ng/mL
RSD (%)
18.0
16.7
15.4
19.6
14.9
Individual
MS
response
compared
to total MS
response
(%)
48.1
31.1
9.6
6.9
4.3
S-4
Average
MS
response
(n = 2)
4091
2887
1538
645
199
15 ng/mL
RSD (%)
5.5
17.7
1.6
12.6
32.7
Individual
MS
response
compared
to total MS
response
(%)
43.7
30.8
16.4
6.9
2.1