Electronic Supplementary Material (ESI) for Chemical Communications.

This journal is © The Royal Society of Chemistry 2014

Supplementary Information for Chemical Communications

Solid phase microextraction (SPME)-transmission mode (TM)

pushes down detection limits in direct analysis in real time (DART)*
Germán Augusto Gómez-Ríos and Janusz Pawliszyn*

Department of Chemistry, University of Waterloo, Waterloo, ON, Canada

Email: janusz@uwaterloo.ca; Fax: 519-746-0435; Tel: 519-888-4567

 Figure S1 Schematic of UW-12 strips SPME-TMDART holder

The holder was developed at the machine shop of the University of Waterloo. It can be used to perform concomitant extractions
on a 96 well-Concept autosampler (PAS technologies [1]), as well as automated and stable desorption/ionizations. The system is
compatible with the automated rail commercialized by IonSense. Up to 12 SPME-TM devices can be easily installed/removed
from the holder, and spatial position can be accurately adjusted on the Z and Y axis.
 Figure S2A. In-Tube Solid-P Phase Microexxtraction with Direct
D Analysis in Real Time M Mass Spectromeetry; image wass adapted
from the originnal source publlished by Wang
g and collaborattors [2]. B. Thin-film solid-ph
hase microextraaction and direcct analysis
in real time; image waas adapted from
m the original so
ource publishedd by Rodriguezz-Lafuente and collaborators [3 3].
 A

Figure S3 Extracttion time prrofiles for A

A. diazepamm and B. coocaine, respectively. Exxtractions
were peerformed uusing a vorttex agitatorr set-up at maximum speed (32000 rpm). Exxtractions
from 1..5 mL of P PBS spikedd with 50 pppb of eachh analyte w with 3 diffeerent bladess (n = 6).
Extractss were anallyzed using Thermo TS SQ LC-MS S/MS on SR RM mode.
 A

Figure S4 A. Quaantitative annalysis of PBS

P spikedd with cocaiine (10 pg mL-1 to 50 ng mL-1)
and its isotopologuue [D3] coccaine (12 ngg mL-1). B. Quantitativve analysiss of PBS sppiked with
diazepaam (10 pg mL-1 to 500 ng mL-1) and its isotopologuee [D5] diazepam (12 nng mL-1).
 RT: 0.12 - 2.59
2
100

95

90

85

80

75

70

65

60
Relative Abundance

55

50

45

40

35

30

25

20

15

10 A
5

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1
1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3
3 2.4 2.5
Tim
me (min)

NL: 2.73E
E2

RT: 0.12 - 2.59 NL: 7.58E5

100

95

90

85

80 0.2 0.3 0.4

0 0.5 0.6 0.7 0.8 0.9 1.0 1..1 1.2 1.3 1.4 1.5 1.6 1.7 1.8
8 1.9 2.0 2.1 2.2 2.3 2.4 2.5
Time (min)
75

70

65

60
Relative Abundance

55

50

45

40

35

30

25

20

15

10

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0

0.9Figure
1.0 1.1 1.2 1.3 1.4 1.5 1.6
6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5
Time (min)
S5 SPMME-TM inteer-mesh repproducibilitty; ion chroonograms oobtained affter 1 min eextraction
from a solution sppiked with 20 ppb of cocaine (ggreen line) vversus A. carry-over
c measured
subsequuently afterr the desorp
rption/ionizaation cyclee (blue linee) and B. carry
c over measured
after clleaning thee SPME-TM M device (red line) on 1.5 m mL of a mixture
m of methanol,
m
isopropanol and accetonitrile ((50:25:25) ffor 30 minuutes.
Figgure S6 Schheme of thee SPME-TM
M mesh-blaade arrangem
ment
 A

Quantitativee analysis oof plasma sspiked withh cocaine (550 pg mL-1 to 50 ng

Figure S7-1 A. Q
mL-1) and
a its isottopologue [D3] cocainne (12 ng mL-1). B. Quantitativve analysiss of urine
spiked with
w cocainne (50 pg mmL-1 to 50 ng mL-1) andd its isotopoologue [D3] cocaine (112 ng mL-
1
).
 A

Figure S7-2 A. Quantitative analysis off plasma sppiked with ddiazepam (5500pg mL-11 to 50 ng
mL-1) aand its isotoopologue [[D5] diazeppam (12 ngg mL-1). B. Quantitativve analysiss of urine
w diazeppam (50 pg mL-1 to 500 ng mL-1) aand its isotoopologue [D
spiked with D5] diazepaam (12 ng
-1
mL ).
 12000
Thousands

y = 206.33x + 19637
R² = 0.998
10000

7..7%, RSD
8000
Peak area counts [au]

6000

4000

2000

A
100.6%, RSD
0
0 10 20 30 40 50
Concentration of Cocain
ne in Urine [ng/m
mL]
6000
Thousands

5000
y = 97.98x + 67693
R² = 0.999

4000 6.8%, RSD

Peak area counts [au]

3000

2000

1000

15..0%, RSD B
0
0 10 20 30 40 50
ncentration DZP in
Con i Urine [ng/mL]
Figure S8 SPME-TM standard free calibration (n=3). A. Quantitative analysis of urine
spiked with cocaine (500 pg mL-1 to 50 ng mL-1) B. Quantitative analysis of urine spiked
with diazepam (500 pg mL-1 to 50 ng mL-1).
 NL: 1.90E5
RT:0.00 - 5.00
MA: 1606220
190000

180000

170000

160000

150000 MA: 1546711

140000 MA: 1568804
4

130000

120000
Intensity

110000

100000

90000

80000

70000

60000

50000

40000

30000

20000 A
10000

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Tiime (min)
RT: 0.00 - 5.00
MA: 655806 MA: 647466 NLL: 9.08E4
90000
MA: 644335
85000

80000

75000

70000

65000

60000
Intensity

55000

50000

45000

40000

35000

30000

25000

20000

B
15000

10000

5000

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3..5 4.0 4.5 5.0
Time (min)

RT: 0.00 - 5.00 6E5

NL: 1.16
MA: 981550
115000

110000
105000

100000 MA: 955254

95000

90000
85000

80000

75000
70000 MA: 853020
Intensity

65000
60000

55000
50000

45000
40000

35000
30000

25000

C
20000

15000
10000

5000
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
5 4.0 4.5 5.0
me (min)
Tim

Figure S9 Ion chrronograms of three controlled subbstances: hheroin (A), ppropranolol (B), and
stanozoolol (C). 1 m
min extracttions were performed using vorttex agitatorr set-up at m
maximum
speed (3200 rpm).. Simultaneeous extracttion from 1.5
1 mL of P PBS spikedd with 20 nng mL-1 of
21 subsstances desscribed on Table S4. Analyses w were perforrmed usingg a Thermoo TSQ on
MRM mode.
  

  A B
 

Figure
F S10 A. SPME-TM connfiguration for individual extraactions and B. SPME-TM 12--strips configuration for high-tthroughput
analysis usinng 96-Concept autosampler (P PAS Technologies) [1].
Table S1-1 Inter- and intra-mesh reproducibility (n=36). Results are reported as ratio of analyte (diazepam) versus internal
standard isotopologue [D5] diazepam. 1 min extractions were performed using vortex agitator set-up at maximum speed (3200
rpm). Extraction from 1.5 mL of PBS spiked with 20 ng mL-1 of each substance. Analyses were performed using Thermo TSQ on
SRM mode. SD, standard deviation. RSD, relative standard deviation.

Experiment Mesh_1 Mesh_2 Mesh_3 Mesh_4 Mesh_5 Mesh_6 Mesh_7 Mesh_8 Mesh_9
Replicate 1 1.91 1.84 1.87 1.77 1.79 1.75 1.75 1.78 1.78
Replicate 2 1.87 1.80 1.77 1.80 1.80 1.80 1.75 1.77 1.71
Replicate 3 1.76 1.84 1.76 1.81 1.77 1.82 1.76 1.83 1.77
Replicate 4 1.80 1.84 1.94 1.86 1.83 1.82 1.77 1.74 1.78
Summary 1.83 1.83 1.84 1.81 1.80 1.80 1.76 1.78 1.76
SD 0.07 0.02 0.09 0.04 0.02 0.04 0.01 0.03 0.03
RSD 3.65 1.15 4.65 2.07 1.36 1.99 0.71 1.93 1.86

Table S1-2 Inter- and intra-mesh reproducibility (n=36). Results are reported as ratio of analyte (cocaine) versus internal
standard isotopologue [D3] cocaine 1 min extractions were performed using vortex agitator set-up at maximum speed (3200
rpm). Extraction from 1.5 mL of PBS spiked with 20 ng mL-1 of each substance. Analyses were performed using Thermo TSQ on
SRM mode. SD, standard deviation. RSD, relative standard deviation.

Experiment Mesh_1 Mesh_2 Mesh_3 Mesh_4 Mesh_5 Mesh_6 Mesh_7 Mesh_8 Mesh_9
Replicate 1 1.48 1.51 1.53 1.62 1.53 1.58 1.59 1.61 1.62
Replicate 2 1.54 1.54 1.59 1.57 1.62 1.58 1.59 1.64 1.59
Replicate 3 1.51 1.50 1.56 1.52 1.57 1.59 1.67 1.55 1.50
Replicate 4 1.55 1.52 1.47 1.54 1.45 1.53 1.61 1.55 1.53
Summary 1.52 1.52 1.54 1.57 1.54 1.57 1.62 1.59 1.56
SD 0.03 0.02 0.05 0.04 0.07 0.03 0.04 0.04 0.05
RSD 2.07 1.00 3.29 2.77 4.69 1.73 2.23 2.76 3.32
Table S2-1 Inter- and intra-mesh carry-over (n=36). Results are reported as ratio of analyte (diazepam) versus internal standard
isotopologue [D5] diazepam. 1 min extractions were performed using vortex agitator set-up at maximum speed (3200 rpm).
Extraction from 1.5 mL of PBS spiked with 20 ng mL-1 of each substance. Analyses were performed using Thermo TSQ on SRM
mode. SD, standard deviation. RSD, relative standard deviation.

Average RSD
Experiment SD % Carryover DART [A2/A1] % Carryover solvent [A2/A1]
[A/Is] [%]
Replicate 1 1.81 0.06 3.15 7.25 0.46
Replicate 2 1.79 0.04 2.40 4.38 0.33
Replicate 3 1.79 0.03 1.93 4.34 0.27
Replicate 4 1.82 0.06 3.10 3.91 0.23
Summary 1.80 0.05 2.71 4.97 0.32

Table S2-2 Inter- and intra-mesh carry-over (n=36). Results are reported as ratio of analyte (cocaine) versus internal standard
isotopologue [D3] cocaine. 1 min extractions were performed using vortex agitator set-up at maximum speed (3200 rpm).
Extraction from 1.5 mL of PBS spiked with 20 ng mL-1 of each substance. Analyses were performed using Thermo TSQ on SRM
mode. SD, standard deviation. RSD, relative standard deviation.

Average RSD
Experiment SD % Carryover DART [A2/A1] % Carryover solvent [A2/A1]
[A/Is] [%]
Replicate 1 1.56 0.05 3.35 3.06 0.50
Replicate 2 1.58 0.03 2.20 1.80 0.11
Replicate 3 1.55 0.05 3.45 2.04 0.06
Replicate 4 1.53 0.05 3.02 2.53 0.06
Summary 1.56 0.05 3.19 2.36 0.18
Table S3 Typical limits of quantification (LOQ) obtained for cocaine using LC-MS/MS, GC-MS and DART-MS/MS with
diverse approaches versus the results presented in this article.

Sample LOQ [ng/mL] Sample Preparation Instrument Authors Reference

Urine 3.5 SPE LC-MS/MS Berg et al. 4
Urine 5 TFME LC-MS/MS Boyaci et al. 5
Urine 0.1 SPME-TM DART-MS/MS Rodríguez-Lafuente et al. 3
Urine 0.002 SPME-TM DART-MS/MS * -
Serum 0.5 on-line SPE LC-MS/MS Bouzas et al. 6
Plasma 25 SPME GC-MS Alvarez et al. 7
Plasma 0.005 SPME-TM DART-MS/MS * -
Table S4 MS/MS parameters used for the analysis of 21 WADA controlled substances in positive mode, as well as instrumental
response of C18-PAN SPME-TM tandem mass spectrometry analysis. Results are based on the integrated peak area obtained for a
20 ng mL-1 solution in PBS. Average peak area (n=3). LOD*, limit of detection estimated.

Parent ion Product ion Collision LOD*

# Compound name Log P S-lenses Polarity
(m/z) (m/z) energy [pg/mL]
1 Amphetamine 1.76 136.099 91.114 17 36 + 112
2 Methamphetamine 2.07 150.112 91.120 19 45 + 20
3 Nikethamide 0.33 179.100 108.102 18 76 + 17
4 Salbutamol 0.64 240.143 148.103 18 59 + 1474
5 Propranolol 3.48 260.123 116.138 17 89 + 31
6 Metoprolol 1.60 268.140 116.146 18 94 + 108
7 Trenbolone 2.27 271.133 165.106 56 97 + 31
8 Clenbuterol 2.61 277.068 203.049 15 70 + 13
9 Testosterone 3.32 289.157 97.123 21 91 + 10
10 Exemestane 3.70 297.173 121.118 19 72 + 17
11 Codeine 1.20 300.105 152.092 64 104 + 46
12 Cocaine 2.30 304.142 182.173 18 87 + 2
13 Bisoprolol 2.14 326.160 116.135 17 102 + 45
14 6-acetylmorphine 0.42 328.126 165.092 37 122 + 21
15 Stanozolol 5.53 329.229 81.108 44 130 + 22
16 Strychnine 1.93 335.155 184.129 36 136 + 33
17 6-acetylcodeine 2.08 342.124 165.092 45 165 + 7
18 Formoterol 2.20 345.133 121.090 32 85 + 831
19 Heroin 1.52 370.133 165.097 48 119 + 13
20 Toremifene 6.80 406.210 72.167 24 108 + 42
21 GW501516 6.29 454.091 257.068 29 108 + 352
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