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 330.2200@hcd28.00 [ 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 330.2200@hcd28.00 [ 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

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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