the detection of designer drugs from plasma via paper ... asms 2017...multiple reaction monitoring...
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Overview Designer drugs are not detected by routine drug screens, and are more potent than traditional drugs
A disposable paper spray cartridge with SPE column can carry out analyte pre-concentration and
ionization
Method optimized for detection of two synthetic cannabinoids JWH-200 and JWH-250
Most frequently abuse synthetic cannabinoids can be detected at sub-ng/mL levels
The Detection of Designer Drugs from Plasma via Paper Spray Mass Spectrometry CartridgeGreta J. Ren and Nicholas Manicke
Department of Chemistry and Chemical Biology, Indiana University
Introduction Designer drugs mimic psychoactive effects of traditional drugs, however, they are typically more
potent and can have unpredictable and severe health effects
They are cheap and marketed as ‘legal highs’, since they cannot be detected by routine drug screens
New (often more dangerous) drugs continue to emerge as known designer drugs become banned
There is a need for a rapid and sensitive analytical method to detect designer drugs
Tetrahydrocannabinol (THC) JWH-250 5F-ADB
Paper spray mass spectrometry can directly analyze biological samples
Advantages: no sample preparation, small sample volume, small solvent volume, no solvent waste,
no carry over, rapid analysis (1-2 minute run), automatable
Cartridge equipped with solid phase extraction (SPE) column can perform analyte pre-concentration
and ionization
SPE helps improve detection limits by allowing larger sample volumes to be used, removing matrix
interferences and pre-concentrating the analytes
Figure 1: Structure of THC, and synthetic cannabinoids JWH-250 and 5F-ADB Figure 2: Cartridge
positioned in front the of
the mass spectrometer inlet
for analysis
Methods Cartridges were made from Delrin® on a
milling machine
Two parts of the cartridge join together via tongue and groove
Bottom part dimensions: 40mm x 26mm x 6 mm (LWH)
Top part dimensions: 14mm x 22mm x 13mm (LWH)
RT: 0.23 - 2.91
0.5 1.0 1.5 2.0 2.5
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
NL: 1.24E7
TIC F: FTMS + p ESI Full ms2 [email protected] [ 50.0000-355.0000] MS Delr in_10ppb_03
Figure 4: Paper spray chronogram with MS/MS in MRM mode,
Thermo Scientific TSQ Vantage
Bottom part Top part SPE Column
Whatman ET 31
SPE material
Nylon membraneAbsorbent pad
Spray substrate
Figure 3: SPE cartridge and column
1. Sample is loaded at the top of the SPE column, and allowed to wick through
2. Water is added to the top of the cartridge to help remove matrix components
3. The cartridge is covered and allowed to dry
4. Cartridge is positioned in front of the MS inlet and spray solvent is added to the top to extract the analytes
5. Voltage is applied to the cartridge, and analyte signal is collected (2-5 minutes)Pro
ce
du
re
Figure 6: Workflow for paper spray analysis with cartridge equipped with SPE1
Bottom part has two separate regions to hold absorbent pad and paper spray substrate
Top part contains the SPE column (3.0 mm Whatman ET31 paper punch, SPE material, 3.0 mm
nylon punch)
Mass spectrometry analysis was performed using Thermo Scientific TSQ Vantage (TSQ) in the
multiple reaction monitoring (MRM) mode and Thermo Scientific Q-Exactive Focus (QE) in the
parallel reaction monitoring (PRM) mode
RT: 0.23 - 2.91
0.5 1.0 1.5 2.0 2.5
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
NL: 1.24E7
TIC F: FTMS + p ESI Full ms2 [email protected] [ 50.0000-355.0000] MS Delr in_10ppb_03
m/z m/z m/z m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
83.0109.0 257.1 328.2
Figure 5: Paper spray chronogram with MS/MS in PRM mode,
Thermo Scientific Q-Exactive Focus
Cannabinoid ISTD Transitions*
JWH-200 AM-2201 d5 m/z 127
385.3 → 77.0
385.3 → 114.1
385.3 → 127.0
385.3 → 155.0
JWH-250 AM-2201 d5 m/z 127
336.281 → 65.1
336.281 → 91.1
336.281 → 121.1
336.281 → 144.1
AM-2201 AM-2201 d5
360.2 → 77.0
360.2 → 127.0
360.2 → 155.0
360.2 → 239.1
AB-CHMINACA AB-CHMINACA d4
357.2→ 145.0
357.2→ 241.1
357.2→ 312.2
357.2→ 340.2
5F-ADBAB-FUBINACA d4
m/z 257.1
378.2 → 145.0
378.2 → 213.1
378.2 → 233.1
378.2 → 318.2
5F-PB-22AB-CHMINACA d4
m/z 149
385.3 → 89.0
385.3 → 116.0
385.3 → 144.0
385.3 → 232.1
XLR-11 AM-2201 d5 m/z 127
330.3 → 55.1
330.3 → 125.1
330.3 → 144.0
330.3 → 232.1
THJ-2201 AM-2201 d5 m/z 127
361.2 → 90.0
361.2 → 145.0
361.2 → 213.1
361.2 → 233.1
Table 1: TSQ MRM transitions, and the ISTD used for
normalization
*Quantitation transition is bolded
Cannabinoid ISTD Transitions
JWH-200 AB-CHMINACA d4 385.3 → 155.0494
JWH-250 AB-CHMINACA d4 336.3→ 121.0652
AM-2201 AB-CHMINACA d4 360.2 → 155.0494
AB-CHMINACA AB-CHMINACA d4 357.2→ 312.2076
5F-ADB AB-FUBINACA d4 378.2 → 251.1193
5F-PB-22 AB-CHMINACA d4 385.3 → 232.1135
XLR-11 AB-CHMINACA d4 330.3 → 125.0966
THJ-2201 AB-FUBINACA d4 361.2 → 251.1193
Table 2: QE PRM transitions, and the ISTD used for normalization
65.06
m/z
91.09
m/z
121.05
m/z
144.05
m/z
0
50
100
0
50
100
0
50
100
0
50
100
65
91
121
144
65.06
m/z
91.09
m/z
121.05
m/z
144.05
m/z
0
50
100
0
50
100
0
50
100
0
50
100
65
91
121
144
50 100 150 200 250 300 350
0
50
100125.0958
232.112385.0287
330.1776269.2658
176.9866
QE and TSQ produced different
fragmentation and different MS/MS spectra
The fragments with the highest intensity were
selected for quantitation
The Detection of Designer Drugs from Plasma via Paper Spray Mass Spectrometry Cartridgeand Nicholas Manicke
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis
Conclusions
References
Acknowledgments Funding from NIH National Institute on Drug Abuse 1R21DA043037-01
Authors also acknowledge funding and other support from Thermo Scientific
1. Zhang, C. & Manicke, N. E. Development of a Paper Spray Mass Spectrometry Cartridge with
Integrated Solid Phase Extraction for Bioanalysis. Anal. Chem. 87, 6212–6219 (2015)
A method was developed and optimized for synthetic cannabinoids JWH-200 and JWH-250
Extraction solvent, SPE sorbent, sample volume, SPE amount and wash steps were investigated
Method was able to detect several synthetic cannabinoids that were most commonly detected in US
toxicology labs in the last two years at sub-ng/mL concentrations
Synthetic cannabinoids can be quantified with the use of an ISTD
The presented method allows for rapid, sensitive (sub ng/mL) detection of synthetic cannabinoids
with minimal sample preparation and no chromatography. 0.0E+00
5.0E+04
1.0E+05
1.5E+05
2.0E+05
2.5E+05
3.0E+05
3.5E+05
4.0E+05
77.0 114.1 127.0 155.0 91.0 121.0 130.0 144.0
JWH-200 JWH-250
Ab
solu
te S
ign
al
Pre-treated Paper Substrate: Blank Plasma
No pre-treatment Sonicated in acetonitrile with 0.1% formic acid
Figure 9: Blank signal for pre-treated and non-treated paper
substrate
0
200
400
600
800
1000
1200
1400
S/
N
Synthetic Cannabinoid
SPE Amount
5 mg 10 mg 20 mg
Figure 10: Comparison between S/N and amount of SPE used
m/z m/z
0
200
400
600
800
1000
1200
1400
1600
S/
N
Synthetic Cannabinoid
Sample Volume
50 µL 100 µL 200 µL
Figure 11: Comparison between S/N and amount of sample loaded
0
0.5
1
1.5
2
2.5
0 2 4 6 8 10
Rati
o (
An
aly
te/
IST
D)
Synthetic Cannabinoid Concentration (ng/mL)
Synthetic Cannabinoids Concentration in Plasma
(TSQ)
JWH-200 JWH-250 AM-2201 AB-CHMINACA
5F-ADB 5F-PB-22 XLR-11 THJ-2201
0
1
2
3
4
5
6
0 2 4 6 8 10
Rati
o (
An
aly
te/
IST
D)
Synthetic Cannabinoid Concentration (ng/mL)
Synthetic Cannabinoids Concentration in Plasma
(QE)
JWH-200 JWH-250 AM-2201 AB-CHMINACA
5F-ADB 5F-PB-22 XLR-11 THJ-2201
Figure 12: Calibration curve for synthetic cannabinoids, MS
analysis performed using TSQFigure 13: Calibration curve for synthetic cannabinoids, MS
analysis performed using QE
Rinsing SPE column with water after loading the sample helps remove matrix components
Washing the paper substrate helps reduce the background signal
For 100 µL of plasma, 10 mg of SPE material gave the best results
Signal to Noise ratio (S/N) increases with larger sample volumes
Optimized method was used to analyze samples on QE
Figure 10: Comparison between S/N and amount of SPE
Cannabinoid
Limit of Detection (LOD) R2
Direct Paper
Spray TSQ
(ng/mL)
SPE TSQ
(ng/mL)
SPE QE
(ng/mL)SPE (TSQ) SPE (QE)
JWH-200 3.0 0.03 0.22 0.9930 0.9872
JWH-250 13.0 0.06 0.14 0.9935 0.9975
AM-2201 5.0 0.014 0.2 0.9989 0.9906
AB-CHMINACA 0.25 0.064 0.08 0.9991 0.9983
5F-ADB 0.3 0.035 0.27 0.9957 0.9904
5F-PB-22 8.5 0.016 0.25 0.9955 0.9840
XLR-11 7.3 0.02 0.15 0.9927 0.9940
THJ-2201 0.5 0.03 0.3 0.9939 0.9751
All synthetic cannabinoids
could be detected sub-
ng/mL levels
Optimized SPE method
decreased the detection
limits ~100 times
Good linearity from 0.1 –
10 ng/mL
Some adjustments may be
necessary to achieve the
same LODs with the QE
Table 4: Limits of detection and R2 obtained from synthetic cannabinoid calibration curves
Results
Figure 8: Analyte signal improvement with the added wash step0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
6.0E+05
7.0E+05
8.0E+05
9.0E+05
1.0E+06
77.0 114.1 127.0 155.0 91.0 121.0 130.0 144.0
JWH-200 JWH-250
Ab
solu
te S
ign
al
Wash Step
0 µL water 50 µL water 300 µL water
m/zm/z
Figure 8: Analyte signal improvement with the added wash step
0.0E+00
2.0E+07
4.0E+07
6.0E+07
8.0E+07
1.0E+08
1.2E+08
1.4E+08
1.6E+08
1.8E+08
2.0E+08
77.0 114.1 127.0 155.0 91.0 121.0 130.0 144.0
JWH-200 JWH-250
Ab
solu
te S
ign
al
Extraction Solvent
95:5 Methanol: Water with 0.01% Acetic AcidMethanol with 0.01% Acetic AcidMethanol with 0.1% Formic Acid50:50 Methanol:Acetonitrile with 0.1% Formic AcidAcetonitrile with 0.1% Formic Acid
m/zm/z
Figure 7: Analyte signal obtained with SPE cartridges with various extraction solvents
SPE MaterialJWH-200
(ng/mL)
JWH-250
(ng/mL)
Strata-X-RP 0.03 0.1
HybridSPE
Phospholipid0.1 1
HLB 0.1 1
SAX 0.1 1
Table 3: Limits of detection using different
SPE materials
Acetonitrile with 0.1% formic acid improved analyte signal ~10x for both analytes
The blank signal was not significantly affected
Solid phase sorbent Strata-X-RP had the lowest detection limits