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Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic ToxicologyMaria C. Prieto Conaway,1 Nicholas E. Manicke,2 Marta Kozak1 1Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134 2Department of Chemistry, Indiana University-Purdue University Indianapolis, IN
2 Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology
Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic ToxicologyMaria C. Prieto Conaway1, Nicholas E. Manicke2, Marta Kozak1
1Thermo Fisher Scientific 355 River Oaks Parkway San Jose CA 95134 2Department of Chemistry Indiana University Purdue University Indianapolis IN1Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose CA 95134, 2Department of Chemistry, Indiana University-Purdue University Indianapolis, IN
Overview FIGURE 6b. Quantitative results for it i t li li d b i t l
FIGURE 6a. Full scan MS spectra for
Purpose: Method development for the rapid and semi-quantitative screening of drugs of abuse in forensic toxicology using paper spray mass spectrometry.
Methods: Bovine blood spiked with common drugs of abuse and analyzed as dried blood spots by paper spray ionization/Orbitrap mass spectrometry. High resolution and accurate
FIGURE 1. Prototype paper spray ion source (Prosolia, Inc., IN) showing, clockwise from top left: paper spray ion source, mechanism for dispensing solvent to the sample, paper cassette indicating sample deposition and DBS-spotted paper cassette electrospraying into mass spectrometer inlet.
ResultsScreening for drugs of abuse: resolving power, accurate mass for compound identification• Figure 2 shows that high and ultrahigh resolving powers (70,000 and 140,000 FWHM
FIGURE 4. Accurate mass (3-4 ppm) MS spectra at 140,000 resolving power (FWHM at m/z 200) showing drugs detected down to 100 ng/mL. Sample contained six drugs analyzed from DBS.
meth-
amitriptyline normalized by internal standard from DBS samples. Calibration curve and %RSD variability (n=3) shown.
the [M+H]+ ion of amitriptyline at various concentrations from DBS samples. Acquired at 70,000 resolving power.
Mass accuracy 2-3 ppmAmitriptyline
Y = -0.0519284+0.001756*X R^2 = 0.9933 W: 1/X
9p y p p p y p p y gmass used in full MS, MS2 and All Ion Fragmentation experiments for the identification and confirmation of drugs from dried blood spot samples. Thermo Scientific™ TraceFinder™ 3.0 software used for data analysis.
Results: Able to identify six drugs of abuse from dried blood spots at a 100 ng/mL level with outstanding signal to noise Limit of detection from dried blood spots with this
g g g g p ( , ,at m/z 200) are required when evaluating samples from complex matrices with no sample preparation and no prior chromatographic separation. Mass accuracies 1-2 ppm at the higher resolving powers (70,000 and 140,000, FWHM at m/z 200).
• Results from TraceFinder software, which is effectively used for targeted or unknown screening analysis, are neatly summarized in Figure 3. All six drugs are positively 40
60
80
100136.1125
40
60
80
100150.1282
150.1313 40
60
80
100244.2067
40
60
80
100300.1604
300.1661
300.1787300.142640
60
80
100304.1552
40
60
80
100318.1710
0 ng
/mL
amphetamine PCP codeine cocaethylenecocainemeth-amphetamine
50
1000
50
100278.1911
278.1910
5000 ng/mL
1000 ng/mL
Mass accuracy 2 3 ppm
3
4
5
6
7
8
9
Are
a R
atio
with outstanding signal to noise. Limit of detection from dried blood spots with this technique is 1-10 ng/mL, compound dependent. Paper spray is easy to use, requires no sample preparation and no prior chromatography, making for a quick technique with the potential to identify compounds in seconds. The Thermo Scientific™ Orbitrap™ Exactive™ family of mass spectrometers are ideally suited for coupling to paper spray ionization.
g y , y g g p yidentified from a dry blood spot sample.
Screening for drugs of abuse at various concentrations• A drug mixture of six compounds was analyzed at 100, 500, 1000 and 2500 ng/mL for
forensic toxicology screening Amphetamine methamphetamine cocaine60
80
1000
20
40
136.1222
136.1125
60
80
100
bund
ance
0
20
40
150.1212
150.1313
150.1282
60
80
100
bund
ance
0
20
40
244.2277
244.1916 244.2475244.2067
244.227860
80
100
bund
ance
0
20
40
300.2024
300.1427
300.1604300.1661
300.1790 60
80
100
bund
ance
0
20
40
304.1910304.1458
304.1646
304.1552
60
80
100
bund
ance
0
20
40
318.1918318.1405
318.2283
318.1708
2500
0 ng
/mL
1000
50
1000
50
278.1910
278.1909
g
500 ng/mL
250 ng/mL
0 1000 2000 3000 4000 5000ng/mL
0
1
2
IntroductionPaper spray is a direct ionization technique that simplifies the mass spectrometric analysis of dried blood spots (DBS). Paper-spray technology is therefore attractive for forensic toxicology screening for drugs of abuse. The sample collection and storage of DBS in a
f f f
forensic toxicology screening. Amphetamine, methamphetamine, cocaine, cocaethylene, codeine and PCP are shown in this work.
• This group of samples were detected by full scan MS down to 100 ng/mL levels (Figure 4) (140,000 resolving power; FWHM at m/z 200).
60
80
1000
20
40
136.1222
136.1247
136.1125
60
80
1000
20
40
Rel
ativ
e A
b
150.1211
150.1282
60
80
1000
20
40
Rel
ativ
e A
b
244.1915 244.2470
244.2067244.2278
60
80
1000
20
40
Rel
ativ
e A
b
300.2024
300.1426
300.1787
300.1603
60
80
1000
20
40
Rel
ativ
e A
b
304.1973
304.1760
304.1552
60
80
1000
20
40
Rel
ativ
e A
b
318.1403318.1919
318.1708
1000
ng/m
L
1000
50
1000
50
278.1910
278.2183
278.1909
250 ng/mL
100 ng/mL
50 ng/mL
Level (ng/mL) % RSD
simple paper cassette make shipment of samples to the forensic toxicology lab safe and convenient. Both qualitative and quantitative analysis of small molecules from complex matrices such as blood or other biological fluids is possible without time consuming sample preparation and chromatography.
Quantitation of DBS samples with paper spray MS is fairly well established even though a
Fragmentation and isotopic pattern matching for compound confirmation• Accurate mass m/z values were used for identification of screened drugs. Isotopic
pattern matching and two fragments from the AIF experiment were used for drug confirmation (TraceFinder table Fig. 3). Alternatively, DD MS/MS from a Q Exactivemass spectrometer can be used. 60
80
1000
20
40
136.1222
136.1247
136.1154
136.1125
60
80
1000
20
40
150.1211
150.1130150.0996
150.1211
150.1130
150.128260
80
1000
20
40
244.1914244.2471
244.2278
244.2066
60
80
1000
20
40
300.2024
300.1426
300.1661 60
80
1000
20
40
304.1891304.1377304.1551
60
80
1000
20
40
318.1404
318.1919
318.1708318.1402
500
ng/m
L
1000
50
1000
50 278.2180
278.1911
278.2182
278.1909278 2180
25 ng/mL
10 ng/mL300.1603
Conclusion
Quantitation of DBS samples with paper-spray MS is fairly well established even though a commercial product is not yet available (1). While previous work used a Thermo Scientific triple quadrupole mass spectrometer and monitored specific MS/MS transitions, full-MS instruments with Orbitrap analyzers are ideally suited as rapid screening tools. Orbitrapanalyzers provide high resolution, accurate mass (HR/AM) analysis for high confidence identification allow for unlimited number of analytes in the method and retrospective data
p• Figure 5 shows accurate mass fragmentation spectra by targeted DD MS/MS for a
DBS sample containing a mixture of 6 drugs. DD MS/MS is acquired at ultra high resolution for enhanced signal to noise. Please note that at the higher resolution, the signal to noise is exceptional thus allowing much lower limits of detection than demonstrated.
136.11 136.12 136.13m/z
0
20
40 136.1206
136.1246
150.10 150.12 150.14m/z
0
20
40
150.0999
150.1363
244.20m/z
0
20
40244.1914
244.2471
300.15 300.20m/z
0
20
40
300.1870
304.15 304.20m/z
0
20
40
304.1763304.1373
318.1 318.2m/z
0
20
40 318.2284
318.1919100
n
278.15 278.20m/z
0
50278.2180g
Conclusion• We have shown an easy to use technique (no sample preparation, no chromatography)
that shows extraordinary potential for the semi-quantitative screening of drugs of abuse in forensic toxicology.
identification, allow for unlimited number of analytes in the method and retrospective data analysis is possible because a full MS spectrum is recorded in addition to All Ion Fragmentation (AIF) or Data Dependent (DD) MS/MS.
In this work, the ability of paper spray coupled to a very sensitive and fast Orbitrapanalyzer is explored for its potential as a forensic toxicology screening tool
demonstrated.
Quantitation• Amitriptyline-spiked in blood (10–5,000 ng/mL) yielded limits of quantitation (LOQ) of
25 ng/mL using amitriptyline-d3 as internal standard (Figure 6).
FIGURE 5. DD MS/MS fragmentation at the highest resolving power of 140,000 (FWHM at m/z 200) in the Q Exactive allows for sensitive detection. An enhanced signal to noise ratio (as compared to MS/MS at 17,500 resolving power, data not shown) is observed. Accurate mass on both precursor and fragments (4-5 ppmand 1-3 ppm, respectively) are used for the identification of compounds in
• Any combination of user required experiments, e.g., MS, AIF and Data Dependent MS/MS, are allowed for the best hit confirmation in a single experiment.
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern
analyzer is explored for its potential as a forensic toxicology screening tool.
MethodsSample Preparation
Mixtures of drugs (Cerilliant TX) were spiked in blood (bovine blood Lampire
FIGURE 2. Full scan MS experiments - highest resolving powers, e.g., 70,000 and 140,000 (FWHM at m/z 200), are required for the identification of drugs from DBS due to matrix interference. Bovine blood spiked with six drugs, four drugs shown below Resolving power from 17 500 35 000 70 000 and 140 000 top to bottom
• Variability in terms of %RSD (Std Dev/Mean*100) is between <1 to 16% for drug in blood. Figure 6 displays amitriptyline data for dried blood spots.
FIGURE 3. TraceFinder 3.0 software results shown below. Data processed in targeted screening analysis mode. All analytes in the mix are positively id tifi d b t / l d fi d b i t i tt d th
screening applications. Sample: mixture of six drugs analyzed from DBS, four shown below. Concentrations noted in each panel.
a) Amphetamine b) Methamphetamine
20
60
100 91.0544
119.0859
150 1280
NL: 1.78E7
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern matching are required to confirm drugs identified solely by accurate mass (Fig. 3).
• We have demonstrated feasibility for rapid blood analysis for intoxication cases where expected concentrations are high (≥100 ng/mL, Fig. 4). DD MS2 data (Fig. 5) indicates
• Mixtures of drugs (Cerilliant, TX) were spiked in blood (bovine blood, LampireBiologicals, New Jersey) stabilized with K2-EDTA. Blood sample integrity maintained by not exceeding 5% of solvent in blood (v/v).
• Twelve microliters of spiked blood sample were loaded to paper cartridges, dried under a nitrogen gas flow for 20 min and loaded into stackers that hold up to 40 cassettes.
below. Resolving power from 17,500, 35,000, 70,000 and 140,000 top to bottom. The [M+H]+ ion is highlighted by a red line. Mass accuracies 1-2 ppm.
identified by exact m/z values and confirmed by isotopic pattern and the presence of two fragments from the AIF experiment (see Table).
Data collected with the Exactive Plus mass spectrometer.60
100 91.0544
119.0858 136 0736
NL: 5.86E61000 ng/mL 1000 ng/mL
a) Amphetamine b) Methamphetamine
20
60
100
20 150.1280
91.0544
119.0859
150.1280
NL: 9.06E6
p g ( g , g ) ( g )lower levels can be achieved and this is part of ongoing investigations.
• High resolution and accurate mass are crucial techniques for analyzing complex samples by MS and nicely complement the paper spray technique in the screening of drugs from d i d bl d t
• Solvent is automatically dispensed to the DBS before analysis and an applied high voltage (3-5 kV) induces electrospray from the sharp tip of the paper (Figure 1).
• The extraction solvent used in this work is 95/5 (v/v) methanol/water with 100 ppm acetic acid (pH 4.5).
244.2284244.2069
300.1592
300.2911
318.1711318.1964
318 13916080
100 136.0738
amphetamine PCP codeine cocaethylene
17 500
RP20
60
100
20119.0858 136.0736
91.0544
136.0736119.0858
NL: 2.68E6
500 ng/mL 500 ng/mL
100 120 1400
20
60
100 149.023791.0544
119.0858
NL: 1.59E6
dried blood spots.
• Data collected in this screening application allows for retrospective analysis as a full scan MS event is always acquired.
Mass Spectrometry• The paper-spray source was coupled to either a Thermo Scientific™ Exactive Plus™ or
a Thermo Scientific™ Q Exactive™ Orbitrap mass spectrometer. • An automated experiment for drug screening consisted of 30 sec data collection,
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and
244.2071
244.2269
300.2190
300.1614
300.1810300.2907
300 2186
318.1391
318.2667318.1716
318.1916
406080
100
tive
Abu
ndan
ce 02040 136.1130
136.0738136.1127
136.1055
17,500
35,00090 100 110 120 130
020
60
100 136.0736
91.0543
119.0857
NL: 1.18E6100 ng/mL 100 ng/mL
60
100 196.1336
82 0653
NL: 6.90E7
100 120 140
60
100 86.0966
159.1173
91 0545
NL: 4.33E7
• The paper spray technique coupled with automated data processing using TraceFinder3.0 software provides a complete solution for drug screening in forensic toxicology.
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and Data Dependent Higher Collision Dissociation (HCD) MS/MS (Q Exactive MS).
• For maximum specificity and sensitivity, both full scan and fragmentation data were acquired at 140,000 resolving power (FWHM at m/z 200). Normalized collision energy was 40 eV.
244.2068
244.2268
300.2186
300.1606
300.2912300.1794
300.1433
318.1432318.2648
318.1715
318.1414 318 2840318.2286
020406080
1000
20
Rel
at
136.1127
136.0739
136.105770,000
Isotopic pattern match
Simulation
90 100 110 120 130
c) PCP d) Cocaethylene
References1000 ng/mL 1000 ng/mL
20
60
100
2082.0653
150.0917 318.1704196.1336
82.0653150 0917
NL: 3.74E7
20
60
100
2091.0545
244.206486.0966
159.1172
91.0544
NL: 2.66E7
• All data acquisition used the Thermo Scientific™ Xcalibur™ sequences and contact closure trigger from the paper spray source.
Data Analysis• Thermo Scientific™ QualBrowser™ software from the Xcalibur platform was used for 244.15 244.20 244.25
244.2066
244.2269
300.1 300.2 300.3
300.1608
300.2910300.2185300.1431
300.3274
318.1 318.2 318.3
318.2840318.1711
318.1417318.1907318.2836136.10
020406080
1000 136.1127
136.0739
136.1056136.0845
140,000
ExperimentalReferences1. Manicke, N.; Yang, Q.; Wang, H.; Oradu, S.; Ouyang, Z.; Cooks, R.G. Assessment of
Paper Spray Ionization for Quantitation of Pharmaceuticals in Blood Spots. IJMS 2011, 300, 123-129.500 ng/mL 500 ng/mL
020
60
100
20 150.0917 318.1705196.1336
80.9480
150.0917105.0337
318.1704
NL: 6.60E6
020
60
100
20 104.1071 244.206386.0966
245.1365159.1172
95.0857135.1171
213.1100
NL: 3.26E6
Thermo Scientific QualBrowser software from the Xcalibur platform was used for spectra visualization. TraceFinder 3.0 software was used for the automated identification and confirmation in the targeted screening of drugs.
For forensic toxicology use only.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others
m/z m/z m/zm/z
100 ng/mL 100 ng/mL
50 100 150 200 250 3000
100 150 200 2500 intellectual property rights of others.
PO64316-EN 1114S
3Thermo Scientific Poster Note • PN64316-EN 1114S
Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic ToxicologyMaria C. Prieto Conaway1, Nicholas E. Manicke2, Marta Kozak1
1Thermo Fisher Scientific 355 River Oaks Parkway San Jose CA 95134 2Department of Chemistry Indiana University Purdue University Indianapolis IN1Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose CA 95134, 2Department of Chemistry, Indiana University-Purdue University Indianapolis, IN
Overview FIGURE 6b. Quantitative results for it i t li li d b i t l
FIGURE 6a. Full scan MS spectra for
Purpose: Method development for the rapid and semi-quantitative screening of drugs of abuse in forensic toxicology using paper spray mass spectrometry.
Methods: Bovine blood spiked with common drugs of abuse and analyzed as dried blood spots by paper spray ionization/Orbitrap mass spectrometry. High resolution and accurate
FIGURE 1. Prototype paper spray ion source (Prosolia, Inc., IN) showing, clockwise from top left: paper spray ion source, mechanism for dispensing solvent to the sample, paper cassette indicating sample deposition and DBS-spotted paper cassette electrospraying into mass spectrometer inlet.
ResultsScreening for drugs of abuse: resolving power, accurate mass for compound identification• Figure 2 shows that high and ultrahigh resolving powers (70,000 and 140,000 FWHM
FIGURE 4. Accurate mass (3-4 ppm) MS spectra at 140,000 resolving power (FWHM at m/z 200) showing drugs detected down to 100 ng/mL. Sample contained six drugs analyzed from DBS.
meth-
amitriptyline normalized by internal standard from DBS samples. Calibration curve and %RSD variability (n=3) shown.
the [M+H]+ ion of amitriptyline at various concentrations from DBS samples. Acquired at 70,000 resolving power.
Mass accuracy 2-3 ppmAmitriptyline
Y = -0.0519284+0.001756*X R^2 = 0.9933 W: 1/X
9p y p p p y p p y gmass used in full MS, MS2 and All Ion Fragmentation experiments for the identification and confirmation of drugs from dried blood spot samples. Thermo Scientific™ TraceFinder™ 3.0 software used for data analysis.
Results: Able to identify six drugs of abuse from dried blood spots at a 100 ng/mL level with outstanding signal to noise Limit of detection from dried blood spots with this
g g g g p ( , ,at m/z 200) are required when evaluating samples from complex matrices with no sample preparation and no prior chromatographic separation. Mass accuracies 1-2 ppm at the higher resolving powers (70,000 and 140,000, FWHM at m/z 200).
• Results from TraceFinder software, which is effectively used for targeted or unknown screening analysis, are neatly summarized in Figure 3. All six drugs are positively 40
60
80
100136.1125
40
60
80
100150.1282
150.1313 40
60
80
100244.2067
40
60
80
100300.1604
300.1661
300.1787300.142640
60
80
100304.1552
40
60
80
100318.1710
0 ng
/mL
amphetamine PCP codeine cocaethylenecocainemeth-amphetamine
50
1000
50
100278.1911
278.1910
5000 ng/mL
1000 ng/mL
Mass accuracy 2 3 ppm
3
4
5
6
7
8
9
Are
a R
atio
with outstanding signal to noise. Limit of detection from dried blood spots with this technique is 1-10 ng/mL, compound dependent. Paper spray is easy to use, requires no sample preparation and no prior chromatography, making for a quick technique with the potential to identify compounds in seconds. The Thermo Scientific™ Orbitrap™ Exactive™ family of mass spectrometers are ideally suited for coupling to paper spray ionization.
g y , y g g p yidentified from a dry blood spot sample.
Screening for drugs of abuse at various concentrations• A drug mixture of six compounds was analyzed at 100, 500, 1000 and 2500 ng/mL for
forensic toxicology screening Amphetamine methamphetamine cocaine60
80
1000
20
40
136.1222
136.1125
60
80
100
bund
ance
0
20
40
150.1212
150.1313
150.1282
60
80
100
bund
ance
0
20
40
244.2277
244.1916 244.2475244.2067
244.227860
80
100
bund
ance
0
20
40
300.2024
300.1427
300.1604300.1661
300.1790 60
80
100
bund
ance
0
20
40
304.1910304.1458
304.1646
304.1552
60
80
100
bund
ance
0
20
40
318.1918318.1405
318.2283
318.1708
2500
0 ng
/mL
1000
50
1000
50
278.1910
278.1909
g
500 ng/mL
250 ng/mL
0 1000 2000 3000 4000 5000ng/mL
0
1
2
IntroductionPaper spray is a direct ionization technique that simplifies the mass spectrometric analysis of dried blood spots (DBS). Paper-spray technology is therefore attractive for forensic toxicology screening for drugs of abuse. The sample collection and storage of DBS in a
f f f
forensic toxicology screening. Amphetamine, methamphetamine, cocaine, cocaethylene, codeine and PCP are shown in this work.
• This group of samples were detected by full scan MS down to 100 ng/mL levels (Figure 4) (140,000 resolving power; FWHM at m/z 200).
60
80
1000
20
40
136.1222
136.1247
136.1125
60
80
1000
20
40
Rel
ativ
e A
b
150.1211
150.1282
60
80
1000
20
40
Rel
ativ
e A
b
244.1915 244.2470
244.2067244.2278
60
80
1000
20
40
Rel
ativ
e A
b
300.2024
300.1426
300.1787
300.1603
60
80
1000
20
40
Rel
ativ
e A
b
304.1973
304.1760
304.1552
60
80
1000
20
40
Rel
ativ
e A
b
318.1403318.1919
318.1708
1000
ng/m
L
1000
50
1000
50
278.1910
278.2183
278.1909
250 ng/mL
100 ng/mL
50 ng/mL
Level (ng/mL) % RSD
simple paper cassette make shipment of samples to the forensic toxicology lab safe and convenient. Both qualitative and quantitative analysis of small molecules from complex matrices such as blood or other biological fluids is possible without time consuming sample preparation and chromatography.
Quantitation of DBS samples with paper spray MS is fairly well established even though a
Fragmentation and isotopic pattern matching for compound confirmation• Accurate mass m/z values were used for identification of screened drugs. Isotopic
pattern matching and two fragments from the AIF experiment were used for drug confirmation (TraceFinder table Fig. 3). Alternatively, DD MS/MS from a Q Exactivemass spectrometer can be used. 60
80
1000
20
40
136.1222
136.1247
136.1154
136.1125
60
80
1000
20
40
150.1211
150.1130150.0996
150.1211
150.1130
150.128260
80
1000
20
40
244.1914244.2471
244.2278
244.2066
60
80
1000
20
40
300.2024
300.1426
300.1661 60
80
1000
20
40
304.1891304.1377304.1551
60
80
1000
20
40
318.1404
318.1919
318.1708318.1402
500
ng/m
L
1000
50
1000
50 278.2180
278.1911
278.2182
278.1909278 2180
25 ng/mL
10 ng/mL300.1603
Conclusion
Quantitation of DBS samples with paper-spray MS is fairly well established even though a commercial product is not yet available (1). While previous work used a Thermo Scientific triple quadrupole mass spectrometer and monitored specific MS/MS transitions, full-MS instruments with Orbitrap analyzers are ideally suited as rapid screening tools. Orbitrapanalyzers provide high resolution, accurate mass (HR/AM) analysis for high confidence identification allow for unlimited number of analytes in the method and retrospective data
p• Figure 5 shows accurate mass fragmentation spectra by targeted DD MS/MS for a
DBS sample containing a mixture of 6 drugs. DD MS/MS is acquired at ultra high resolution for enhanced signal to noise. Please note that at the higher resolution, the signal to noise is exceptional thus allowing much lower limits of detection than demonstrated.
136.11 136.12 136.13m/z
0
20
40 136.1206
136.1246
150.10 150.12 150.14m/z
0
20
40
150.0999
150.1363
244.20m/z
0
20
40244.1914
244.2471
300.15 300.20m/z
0
20
40
300.1870
304.15 304.20m/z
0
20
40
304.1763304.1373
318.1 318.2m/z
0
20
40 318.2284
318.1919100
n
278.15 278.20m/z
0
50278.2180g
Conclusion• We have shown an easy to use technique (no sample preparation, no chromatography)
that shows extraordinary potential for the semi-quantitative screening of drugs of abuse in forensic toxicology.
identification, allow for unlimited number of analytes in the method and retrospective data analysis is possible because a full MS spectrum is recorded in addition to All Ion Fragmentation (AIF) or Data Dependent (DD) MS/MS.
In this work, the ability of paper spray coupled to a very sensitive and fast Orbitrapanalyzer is explored for its potential as a forensic toxicology screening tool
demonstrated.
Quantitation• Amitriptyline-spiked in blood (10–5,000 ng/mL) yielded limits of quantitation (LOQ) of
25 ng/mL using amitriptyline-d3 as internal standard (Figure 6).
FIGURE 5. DD MS/MS fragmentation at the highest resolving power of 140,000 (FWHM at m/z 200) in the Q Exactive allows for sensitive detection. An enhanced signal to noise ratio (as compared to MS/MS at 17,500 resolving power, data not shown) is observed. Accurate mass on both precursor and fragments (4-5 ppmand 1-3 ppm, respectively) are used for the identification of compounds in
• Any combination of user required experiments, e.g., MS, AIF and Data Dependent MS/MS, are allowed for the best hit confirmation in a single experiment.
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern
analyzer is explored for its potential as a forensic toxicology screening tool.
MethodsSample Preparation
Mixtures of drugs (Cerilliant TX) were spiked in blood (bovine blood Lampire
FIGURE 2. Full scan MS experiments - highest resolving powers, e.g., 70,000 and 140,000 (FWHM at m/z 200), are required for the identification of drugs from DBS due to matrix interference. Bovine blood spiked with six drugs, four drugs shown below Resolving power from 17 500 35 000 70 000 and 140 000 top to bottom
• Variability in terms of %RSD (Std Dev/Mean*100) is between <1 to 16% for drug in blood. Figure 6 displays amitriptyline data for dried blood spots.
FIGURE 3. TraceFinder 3.0 software results shown below. Data processed in targeted screening analysis mode. All analytes in the mix are positively id tifi d b t / l d fi d b i t i tt d th
screening applications. Sample: mixture of six drugs analyzed from DBS, four shown below. Concentrations noted in each panel.
a) Amphetamine b) Methamphetamine
20
60
100 91.0544
119.0859
150 1280
NL: 1.78E7
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern matching are required to confirm drugs identified solely by accurate mass (Fig. 3).
• We have demonstrated feasibility for rapid blood analysis for intoxication cases where expected concentrations are high (≥100 ng/mL, Fig. 4). DD MS2 data (Fig. 5) indicates
• Mixtures of drugs (Cerilliant, TX) were spiked in blood (bovine blood, LampireBiologicals, New Jersey) stabilized with K2-EDTA. Blood sample integrity maintained by not exceeding 5% of solvent in blood (v/v).
• Twelve microliters of spiked blood sample were loaded to paper cartridges, dried under a nitrogen gas flow for 20 min and loaded into stackers that hold up to 40 cassettes.
below. Resolving power from 17,500, 35,000, 70,000 and 140,000 top to bottom. The [M+H]+ ion is highlighted by a red line. Mass accuracies 1-2 ppm.
identified by exact m/z values and confirmed by isotopic pattern and the presence of two fragments from the AIF experiment (see Table).
Data collected with the Exactive Plus mass spectrometer.60
100 91.0544
119.0858 136 0736
NL: 5.86E61000 ng/mL 1000 ng/mL
a) Amphetamine b) Methamphetamine
20
60
100
20 150.1280
91.0544
119.0859
150.1280
NL: 9.06E6
p g ( g , g ) ( g )lower levels can be achieved and this is part of ongoing investigations.
• High resolution and accurate mass are crucial techniques for analyzing complex samples by MS and nicely complement the paper spray technique in the screening of drugs from d i d bl d t
• Solvent is automatically dispensed to the DBS before analysis and an applied high voltage (3-5 kV) induces electrospray from the sharp tip of the paper (Figure 1).
• The extraction solvent used in this work is 95/5 (v/v) methanol/water with 100 ppm acetic acid (pH 4.5).
244.2284244.2069
300.1592
300.2911
318.1711318.1964
318 13916080
100 136.0738
amphetamine PCP codeine cocaethylene
17 500
RP20
60
100
20119.0858 136.0736
91.0544
136.0736119.0858
NL: 2.68E6
500 ng/mL 500 ng/mL
100 120 1400
20
60
100 149.023791.0544
119.0858
NL: 1.59E6
dried blood spots.
• Data collected in this screening application allows for retrospective analysis as a full scan MS event is always acquired.
Mass Spectrometry• The paper-spray source was coupled to either a Thermo Scientific™ Exactive Plus™ or
a Thermo Scientific™ Q Exactive™ Orbitrap mass spectrometer. • An automated experiment for drug screening consisted of 30 sec data collection,
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and
244.2071
244.2269
300.2190
300.1614
300.1810300.2907
300 2186
318.1391
318.2667318.1716
318.1916
406080
100
tive
Abu
ndan
ce 02040 136.1130
136.0738136.1127
136.1055
17,500
35,00090 100 110 120 130
020
60
100 136.0736
91.0543
119.0857
NL: 1.18E6100 ng/mL 100 ng/mL
60
100 196.1336
82 0653
NL: 6.90E7
100 120 140
60
100 86.0966
159.1173
91 0545
NL: 4.33E7
• The paper spray technique coupled with automated data processing using TraceFinder3.0 software provides a complete solution for drug screening in forensic toxicology.
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and Data Dependent Higher Collision Dissociation (HCD) MS/MS (Q Exactive MS).
• For maximum specificity and sensitivity, both full scan and fragmentation data were acquired at 140,000 resolving power (FWHM at m/z 200). Normalized collision energy was 40 eV.
244.2068
244.2268
300.2186
300.1606
300.2912300.1794
300.1433
318.1432318.2648
318.1715
318.1414 318 2840318.2286
020406080
1000
20
Rel
at
136.1127
136.0739
136.105770,000
Isotopic pattern match
Simulation
90 100 110 120 130
c) PCP d) Cocaethylene
References1000 ng/mL 1000 ng/mL
20
60
100
2082.0653
150.0917 318.1704196.1336
82.0653150 0917
NL: 3.74E7
20
60
100
2091.0545
244.206486.0966
159.1172
91.0544
NL: 2.66E7
• All data acquisition used the Thermo Scientific™ Xcalibur™ sequences and contact closure trigger from the paper spray source.
Data Analysis• Thermo Scientific™ QualBrowser™ software from the Xcalibur platform was used for 244.15 244.20 244.25
244.2066
244.2269
300.1 300.2 300.3
300.1608
300.2910300.2185300.1431
300.3274
318.1 318.2 318.3
318.2840318.1711
318.1417318.1907318.2836136.10
020406080
1000 136.1127
136.0739
136.1056136.0845
140,000
ExperimentalReferences1. Manicke, N.; Yang, Q.; Wang, H.; Oradu, S.; Ouyang, Z.; Cooks, R.G. Assessment of
Paper Spray Ionization for Quantitation of Pharmaceuticals in Blood Spots. IJMS 2011, 300, 123-129.500 ng/mL 500 ng/mL
020
60
100
20 150.0917 318.1705196.1336
80.9480
150.0917105.0337
318.1704
NL: 6.60E6
020
60
100
20 104.1071 244.206386.0966
245.1365159.1172
95.0857135.1171
213.1100
NL: 3.26E6
Thermo Scientific QualBrowser software from the Xcalibur platform was used for spectra visualization. TraceFinder 3.0 software was used for the automated identification and confirmation in the targeted screening of drugs.
For forensic toxicology use only.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others
m/z m/z m/zm/z
100 ng/mL 100 ng/mL
50 100 150 200 250 3000
100 150 200 2500 intellectual property rights of others.
PO64316-EN 1114S
4 Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology
Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic ToxicologyMaria C. Prieto Conaway1, Nicholas E. Manicke2, Marta Kozak1
1Thermo Fisher Scientific 355 River Oaks Parkway San Jose CA 95134 2Department of Chemistry Indiana University Purdue University Indianapolis IN1Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose CA 95134, 2Department of Chemistry, Indiana University-Purdue University Indianapolis, IN
Overview FIGURE 6b. Quantitative results for it i t li li d b i t l
FIGURE 6a. Full scan MS spectra for
Purpose: Method development for the rapid and semi-quantitative screening of drugs of abuse in forensic toxicology using paper spray mass spectrometry.
Methods: Bovine blood spiked with common drugs of abuse and analyzed as dried blood spots by paper spray ionization/Orbitrap mass spectrometry. High resolution and accurate
FIGURE 1. Prototype paper spray ion source (Prosolia, Inc., IN) showing, clockwise from top left: paper spray ion source, mechanism for dispensing solvent to the sample, paper cassette indicating sample deposition and DBS-spotted paper cassette electrospraying into mass spectrometer inlet.
ResultsScreening for drugs of abuse: resolving power, accurate mass for compound identification• Figure 2 shows that high and ultrahigh resolving powers (70,000 and 140,000 FWHM
FIGURE 4. Accurate mass (3-4 ppm) MS spectra at 140,000 resolving power (FWHM at m/z 200) showing drugs detected down to 100 ng/mL. Sample contained six drugs analyzed from DBS.
meth-
amitriptyline normalized by internal standard from DBS samples. Calibration curve and %RSD variability (n=3) shown.
the [M+H]+ ion of amitriptyline at various concentrations from DBS samples. Acquired at 70,000 resolving power.
Mass accuracy 2-3 ppmAmitriptyline
Y = -0.0519284+0.001756*X R^2 = 0.9933 W: 1/X
9p y p p p y p p y gmass used in full MS, MS2 and All Ion Fragmentation experiments for the identification and confirmation of drugs from dried blood spot samples. Thermo Scientific™ TraceFinder™ 3.0 software used for data analysis.
Results: Able to identify six drugs of abuse from dried blood spots at a 100 ng/mL level with outstanding signal to noise Limit of detection from dried blood spots with this
g g g g p ( , ,at m/z 200) are required when evaluating samples from complex matrices with no sample preparation and no prior chromatographic separation. Mass accuracies 1-2 ppm at the higher resolving powers (70,000 and 140,000, FWHM at m/z 200).
• Results from TraceFinder software, which is effectively used for targeted or unknown screening analysis, are neatly summarized in Figure 3. All six drugs are positively 40
60
80
100136.1125
40
60
80
100150.1282
150.1313 40
60
80
100244.2067
40
60
80
100300.1604
300.1661
300.1787300.142640
60
80
100304.1552
40
60
80
100318.1710
0 ng
/mL
amphetamine PCP codeine cocaethylenecocainemeth-amphetamine
50
1000
50
100278.1911
278.1910
5000 ng/mL
1000 ng/mL
Mass accuracy 2 3 ppm
3
4
5
6
7
8
9
Are
a R
atio
with outstanding signal to noise. Limit of detection from dried blood spots with this technique is 1-10 ng/mL, compound dependent. Paper spray is easy to use, requires no sample preparation and no prior chromatography, making for a quick technique with the potential to identify compounds in seconds. The Thermo Scientific™ Orbitrap™ Exactive™ family of mass spectrometers are ideally suited for coupling to paper spray ionization.
g y , y g g p yidentified from a dry blood spot sample.
Screening for drugs of abuse at various concentrations• A drug mixture of six compounds was analyzed at 100, 500, 1000 and 2500 ng/mL for
forensic toxicology screening Amphetamine methamphetamine cocaine60
80
1000
20
40
136.1222
136.1125
60
80
100
bund
ance
0
20
40
150.1212
150.1313
150.1282
60
80
100
bund
ance
0
20
40
244.2277
244.1916 244.2475244.2067
244.227860
80
100
bund
ance
0
20
40
300.2024
300.1427
300.1604300.1661
300.1790 60
80
100
bund
ance
0
20
40
304.1910304.1458
304.1646
304.1552
60
80
100
bund
ance
0
20
40
318.1918318.1405
318.2283
318.1708
2500
0 ng
/mL
1000
50
1000
50
278.1910
278.1909
g
500 ng/mL
250 ng/mL
0 1000 2000 3000 4000 5000ng/mL
0
1
2
IntroductionPaper spray is a direct ionization technique that simplifies the mass spectrometric analysis of dried blood spots (DBS). Paper-spray technology is therefore attractive for forensic toxicology screening for drugs of abuse. The sample collection and storage of DBS in a
f f f
forensic toxicology screening. Amphetamine, methamphetamine, cocaine, cocaethylene, codeine and PCP are shown in this work.
• This group of samples were detected by full scan MS down to 100 ng/mL levels (Figure 4) (140,000 resolving power; FWHM at m/z 200).
60
80
1000
20
40
136.1222
136.1247
136.1125
60
80
1000
20
40
Rel
ativ
e A
b
150.1211
150.1282
60
80
1000
20
40
Rel
ativ
e A
b
244.1915 244.2470
244.2067244.2278
60
80
1000
20
40
Rel
ativ
e A
b
300.2024
300.1426
300.1787
300.1603
60
80
1000
20
40
Rel
ativ
e A
b
304.1973
304.1760
304.1552
60
80
1000
20
40
Rel
ativ
e A
b
318.1403318.1919
318.1708
1000
ng/m
L
1000
50
1000
50
278.1910
278.2183
278.1909
250 ng/mL
100 ng/mL
50 ng/mL
Level (ng/mL) % RSD
simple paper cassette make shipment of samples to the forensic toxicology lab safe and convenient. Both qualitative and quantitative analysis of small molecules from complex matrices such as blood or other biological fluids is possible without time consuming sample preparation and chromatography.
Quantitation of DBS samples with paper spray MS is fairly well established even though a
Fragmentation and isotopic pattern matching for compound confirmation• Accurate mass m/z values were used for identification of screened drugs. Isotopic
pattern matching and two fragments from the AIF experiment were used for drug confirmation (TraceFinder table Fig. 3). Alternatively, DD MS/MS from a Q Exactivemass spectrometer can be used. 60
80
1000
20
40
136.1222
136.1247
136.1154
136.1125
60
80
1000
20
40
150.1211
150.1130150.0996
150.1211
150.1130
150.128260
80
1000
20
40
244.1914244.2471
244.2278
244.2066
60
80
1000
20
40
300.2024
300.1426
300.1661 60
80
1000
20
40
304.1891304.1377304.1551
60
80
1000
20
40
318.1404
318.1919
318.1708318.1402
500
ng/m
L
1000
50
1000
50 278.2180
278.1911
278.2182
278.1909278 2180
25 ng/mL
10 ng/mL300.1603
Conclusion
Quantitation of DBS samples with paper-spray MS is fairly well established even though a commercial product is not yet available (1). While previous work used a Thermo Scientific triple quadrupole mass spectrometer and monitored specific MS/MS transitions, full-MS instruments with Orbitrap analyzers are ideally suited as rapid screening tools. Orbitrapanalyzers provide high resolution, accurate mass (HR/AM) analysis for high confidence identification allow for unlimited number of analytes in the method and retrospective data
p• Figure 5 shows accurate mass fragmentation spectra by targeted DD MS/MS for a
DBS sample containing a mixture of 6 drugs. DD MS/MS is acquired at ultra high resolution for enhanced signal to noise. Please note that at the higher resolution, the signal to noise is exceptional thus allowing much lower limits of detection than demonstrated.
136.11 136.12 136.13m/z
0
20
40 136.1206
136.1246
150.10 150.12 150.14m/z
0
20
40
150.0999
150.1363
244.20m/z
0
20
40244.1914
244.2471
300.15 300.20m/z
0
20
40
300.1870
304.15 304.20m/z
0
20
40
304.1763304.1373
318.1 318.2m/z
0
20
40 318.2284
318.1919100
n
278.15 278.20m/z
0
50278.2180g
Conclusion• We have shown an easy to use technique (no sample preparation, no chromatography)
that shows extraordinary potential for the semi-quantitative screening of drugs of abuse in forensic toxicology.
identification, allow for unlimited number of analytes in the method and retrospective data analysis is possible because a full MS spectrum is recorded in addition to All Ion Fragmentation (AIF) or Data Dependent (DD) MS/MS.
In this work, the ability of paper spray coupled to a very sensitive and fast Orbitrapanalyzer is explored for its potential as a forensic toxicology screening tool
demonstrated.
Quantitation• Amitriptyline-spiked in blood (10–5,000 ng/mL) yielded limits of quantitation (LOQ) of
25 ng/mL using amitriptyline-d3 as internal standard (Figure 6).
FIGURE 5. DD MS/MS fragmentation at the highest resolving power of 140,000 (FWHM at m/z 200) in the Q Exactive allows for sensitive detection. An enhanced signal to noise ratio (as compared to MS/MS at 17,500 resolving power, data not shown) is observed. Accurate mass on both precursor and fragments (4-5 ppmand 1-3 ppm, respectively) are used for the identification of compounds in
• Any combination of user required experiments, e.g., MS, AIF and Data Dependent MS/MS, are allowed for the best hit confirmation in a single experiment.
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern
analyzer is explored for its potential as a forensic toxicology screening tool.
MethodsSample Preparation
Mixtures of drugs (Cerilliant TX) were spiked in blood (bovine blood Lampire
FIGURE 2. Full scan MS experiments - highest resolving powers, e.g., 70,000 and 140,000 (FWHM at m/z 200), are required for the identification of drugs from DBS due to matrix interference. Bovine blood spiked with six drugs, four drugs shown below Resolving power from 17 500 35 000 70 000 and 140 000 top to bottom
• Variability in terms of %RSD (Std Dev/Mean*100) is between <1 to 16% for drug in blood. Figure 6 displays amitriptyline data for dried blood spots.
FIGURE 3. TraceFinder 3.0 software results shown below. Data processed in targeted screening analysis mode. All analytes in the mix are positively id tifi d b t / l d fi d b i t i tt d th
screening applications. Sample: mixture of six drugs analyzed from DBS, four shown below. Concentrations noted in each panel.
a) Amphetamine b) Methamphetamine
20
60
100 91.0544
119.0859
150 1280
NL: 1.78E7
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern matching are required to confirm drugs identified solely by accurate mass (Fig. 3).
• We have demonstrated feasibility for rapid blood analysis for intoxication cases where expected concentrations are high (≥100 ng/mL, Fig. 4). DD MS2 data (Fig. 5) indicates
• Mixtures of drugs (Cerilliant, TX) were spiked in blood (bovine blood, LampireBiologicals, New Jersey) stabilized with K2-EDTA. Blood sample integrity maintained by not exceeding 5% of solvent in blood (v/v).
• Twelve microliters of spiked blood sample were loaded to paper cartridges, dried under a nitrogen gas flow for 20 min and loaded into stackers that hold up to 40 cassettes.
below. Resolving power from 17,500, 35,000, 70,000 and 140,000 top to bottom. The [M+H]+ ion is highlighted by a red line. Mass accuracies 1-2 ppm.
identified by exact m/z values and confirmed by isotopic pattern and the presence of two fragments from the AIF experiment (see Table).
Data collected with the Exactive Plus mass spectrometer.60
100 91.0544
119.0858 136 0736
NL: 5.86E61000 ng/mL 1000 ng/mL
a) Amphetamine b) Methamphetamine
20
60
100
20 150.1280
91.0544
119.0859
150.1280
NL: 9.06E6
p g ( g , g ) ( g )lower levels can be achieved and this is part of ongoing investigations.
• High resolution and accurate mass are crucial techniques for analyzing complex samples by MS and nicely complement the paper spray technique in the screening of drugs from d i d bl d t
• Solvent is automatically dispensed to the DBS before analysis and an applied high voltage (3-5 kV) induces electrospray from the sharp tip of the paper (Figure 1).
• The extraction solvent used in this work is 95/5 (v/v) methanol/water with 100 ppm acetic acid (pH 4.5).
244.2284244.2069
300.1592
300.2911
318.1711318.1964
318 13916080
100 136.0738
amphetamine PCP codeine cocaethylene
17 500
RP20
60
100
20119.0858 136.0736
91.0544
136.0736119.0858
NL: 2.68E6
500 ng/mL 500 ng/mL
100 120 1400
20
60
100 149.023791.0544
119.0858
NL: 1.59E6
dried blood spots.
• Data collected in this screening application allows for retrospective analysis as a full scan MS event is always acquired.
Mass Spectrometry• The paper-spray source was coupled to either a Thermo Scientific™ Exactive Plus™ or
a Thermo Scientific™ Q Exactive™ Orbitrap mass spectrometer. • An automated experiment for drug screening consisted of 30 sec data collection,
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and
244.2071
244.2269
300.2190
300.1614
300.1810300.2907
300 2186
318.1391
318.2667318.1716
318.1916
406080
100
tive
Abu
ndan
ce 02040 136.1130
136.0738136.1127
136.1055
17,500
35,00090 100 110 120 130
020
60
100 136.0736
91.0543
119.0857
NL: 1.18E6100 ng/mL 100 ng/mL
60
100 196.1336
82 0653
NL: 6.90E7
100 120 140
60
100 86.0966
159.1173
91 0545
NL: 4.33E7
• The paper spray technique coupled with automated data processing using TraceFinder3.0 software provides a complete solution for drug screening in forensic toxicology.
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and Data Dependent Higher Collision Dissociation (HCD) MS/MS (Q Exactive MS).
• For maximum specificity and sensitivity, both full scan and fragmentation data were acquired at 140,000 resolving power (FWHM at m/z 200). Normalized collision energy was 40 eV.
244.2068
244.2268
300.2186
300.1606
300.2912300.1794
300.1433
318.1432318.2648
318.1715
318.1414 318 2840318.2286
020406080
1000
20
Rel
at
136.1127
136.0739
136.105770,000
Isotopic pattern match
Simulation
90 100 110 120 130
c) PCP d) Cocaethylene
References1000 ng/mL 1000 ng/mL
20
60
100
2082.0653
150.0917 318.1704196.1336
82.0653150 0917
NL: 3.74E7
20
60
100
2091.0545
244.206486.0966
159.1172
91.0544
NL: 2.66E7
• All data acquisition used the Thermo Scientific™ Xcalibur™ sequences and contact closure trigger from the paper spray source.
Data Analysis• Thermo Scientific™ QualBrowser™ software from the Xcalibur platform was used for 244.15 244.20 244.25
244.2066
244.2269
300.1 300.2 300.3
300.1608
300.2910300.2185300.1431
300.3274
318.1 318.2 318.3
318.2840318.1711
318.1417318.1907318.2836136.10
020406080
1000 136.1127
136.0739
136.1056136.0845
140,000
ExperimentalReferences1. Manicke, N.; Yang, Q.; Wang, H.; Oradu, S.; Ouyang, Z.; Cooks, R.G. Assessment of
Paper Spray Ionization for Quantitation of Pharmaceuticals in Blood Spots. IJMS 2011, 300, 123-129.500 ng/mL 500 ng/mL
020
60
100
20 150.0917 318.1705196.1336
80.9480
150.0917105.0337
318.1704
NL: 6.60E6
020
60
100
20 104.1071 244.206386.0966
245.1365159.1172
95.0857135.1171
213.1100
NL: 3.26E6
Thermo Scientific QualBrowser software from the Xcalibur platform was used for spectra visualization. TraceFinder 3.0 software was used for the automated identification and confirmation in the targeted screening of drugs.
For forensic toxicology use only.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others
m/z m/z m/zm/z
100 ng/mL 100 ng/mL
50 100 150 200 250 3000
100 150 200 2500 intellectual property rights of others.
PO64316-EN 1114S
5Thermo Scientific Poster Note • PN64316-EN 1114S
Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic ToxicologyMaria C. Prieto Conaway1, Nicholas E. Manicke2, Marta Kozak1
1Thermo Fisher Scientific 355 River Oaks Parkway San Jose CA 95134 2Department of Chemistry Indiana University Purdue University Indianapolis IN1Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose CA 95134, 2Department of Chemistry, Indiana University-Purdue University Indianapolis, IN
Overview FIGURE 6b. Quantitative results for it i t li li d b i t l
FIGURE 6a. Full scan MS spectra for
Purpose: Method development for the rapid and semi-quantitative screening of drugs of abuse in forensic toxicology using paper spray mass spectrometry.
Methods: Bovine blood spiked with common drugs of abuse and analyzed as dried blood spots by paper spray ionization/Orbitrap mass spectrometry. High resolution and accurate
FIGURE 1. Prototype paper spray ion source (Prosolia, Inc., IN) showing, clockwise from top left: paper spray ion source, mechanism for dispensing solvent to the sample, paper cassette indicating sample deposition and DBS-spotted paper cassette electrospraying into mass spectrometer inlet.
ResultsScreening for drugs of abuse: resolving power, accurate mass for compound identification• Figure 2 shows that high and ultrahigh resolving powers (70,000 and 140,000 FWHM
FIGURE 4. Accurate mass (3-4 ppm) MS spectra at 140,000 resolving power (FWHM at m/z 200) showing drugs detected down to 100 ng/mL. Sample contained six drugs analyzed from DBS.
meth-
amitriptyline normalized by internal standard from DBS samples. Calibration curve and %RSD variability (n=3) shown.
the [M+H]+ ion of amitriptyline at various concentrations from DBS samples. Acquired at 70,000 resolving power.
Mass accuracy 2-3 ppmAmitriptyline
Y = -0.0519284+0.001756*X R^2 = 0.9933 W: 1/X
9p y p p p y p p y gmass used in full MS, MS2 and All Ion Fragmentation experiments for the identification and confirmation of drugs from dried blood spot samples. Thermo Scientific™ TraceFinder™ 3.0 software used for data analysis.
Results: Able to identify six drugs of abuse from dried blood spots at a 100 ng/mL level with outstanding signal to noise Limit of detection from dried blood spots with this
g g g g p ( , ,at m/z 200) are required when evaluating samples from complex matrices with no sample preparation and no prior chromatographic separation. Mass accuracies 1-2 ppm at the higher resolving powers (70,000 and 140,000, FWHM at m/z 200).
• Results from TraceFinder software, which is effectively used for targeted or unknown screening analysis, are neatly summarized in Figure 3. All six drugs are positively 40
60
80
100136.1125
40
60
80
100150.1282
150.1313 40
60
80
100244.2067
40
60
80
100300.1604
300.1661
300.1787300.142640
60
80
100304.1552
40
60
80
100318.1710
0 ng
/mL
amphetamine PCP codeine cocaethylenecocainemeth-amphetamine
50
1000
50
100278.1911
278.1910
5000 ng/mL
1000 ng/mL
Mass accuracy 2 3 ppm
3
4
5
6
7
8
9
Are
a R
atio
with outstanding signal to noise. Limit of detection from dried blood spots with this technique is 1-10 ng/mL, compound dependent. Paper spray is easy to use, requires no sample preparation and no prior chromatography, making for a quick technique with the potential to identify compounds in seconds. The Thermo Scientific™ Orbitrap™ Exactive™ family of mass spectrometers are ideally suited for coupling to paper spray ionization.
g y , y g g p yidentified from a dry blood spot sample.
Screening for drugs of abuse at various concentrations• A drug mixture of six compounds was analyzed at 100, 500, 1000 and 2500 ng/mL for
forensic toxicology screening Amphetamine methamphetamine cocaine60
80
1000
20
40
136.1222
136.1125
60
80
100
bund
ance
0
20
40
150.1212
150.1313
150.1282
60
80
100
bund
ance
0
20
40
244.2277
244.1916 244.2475244.2067
244.227860
80
100
bund
ance
0
20
40
300.2024
300.1427
300.1604300.1661
300.1790 60
80
100
bund
ance
0
20
40
304.1910304.1458
304.1646
304.1552
60
80
100
bund
ance
0
20
40
318.1918318.1405
318.2283
318.1708
2500
0 ng
/mL
1000
50
1000
50
278.1910
278.1909
g
500 ng/mL
250 ng/mL
0 1000 2000 3000 4000 5000ng/mL
0
1
2
IntroductionPaper spray is a direct ionization technique that simplifies the mass spectrometric analysis of dried blood spots (DBS). Paper-spray technology is therefore attractive for forensic toxicology screening for drugs of abuse. The sample collection and storage of DBS in a
f f f
forensic toxicology screening. Amphetamine, methamphetamine, cocaine, cocaethylene, codeine and PCP are shown in this work.
• This group of samples were detected by full scan MS down to 100 ng/mL levels (Figure 4) (140,000 resolving power; FWHM at m/z 200).
60
80
1000
20
40
136.1222
136.1247
136.1125
60
80
1000
20
40
Rel
ativ
e A
b150.1211
150.1282
60
80
1000
20
40
Rel
ativ
e A
b
244.1915 244.2470
244.2067244.2278
60
80
1000
20
40
Rel
ativ
e A
b
300.2024
300.1426
300.1787
300.1603
60
80
1000
20
40
Rel
ativ
e A
b
304.1973
304.1760
304.1552
60
80
1000
20
40
Rel
ativ
e A
b
318.1403318.1919
318.1708
1000
ng/m
L
1000
50
1000
50
278.1910
278.2183
278.1909
250 ng/mL
100 ng/mL
50 ng/mL
Level (ng/mL) % RSD
simple paper cassette make shipment of samples to the forensic toxicology lab safe and convenient. Both qualitative and quantitative analysis of small molecules from complex matrices such as blood or other biological fluids is possible without time consuming sample preparation and chromatography.
Quantitation of DBS samples with paper spray MS is fairly well established even though a
Fragmentation and isotopic pattern matching for compound confirmation• Accurate mass m/z values were used for identification of screened drugs. Isotopic
pattern matching and two fragments from the AIF experiment were used for drug confirmation (TraceFinder table Fig. 3). Alternatively, DD MS/MS from a Q Exactivemass spectrometer can be used. 60
80
1000
20
40
136.1222
136.1247
136.1154
136.1125
60
80
1000
20
40
150.1211
150.1130150.0996
150.1211
150.1130
150.128260
80
1000
20
40
244.1914244.2471
244.2278
244.2066
60
80
1000
20
40
300.2024
300.1426
300.1661 60
80
1000
20
40
304.1891304.1377304.1551
60
80
1000
20
40
318.1404
318.1919
318.1708318.1402
500
ng/m
L
1000
50
1000
50 278.2180
278.1911
278.2182
278.1909278 2180
25 ng/mL
10 ng/mL300.1603
Conclusion
Quantitation of DBS samples with paper-spray MS is fairly well established even though a commercial product is not yet available (1). While previous work used a Thermo Scientific triple quadrupole mass spectrometer and monitored specific MS/MS transitions, full-MS instruments with Orbitrap analyzers are ideally suited as rapid screening tools. Orbitrapanalyzers provide high resolution, accurate mass (HR/AM) analysis for high confidence identification allow for unlimited number of analytes in the method and retrospective data
p• Figure 5 shows accurate mass fragmentation spectra by targeted DD MS/MS for a
DBS sample containing a mixture of 6 drugs. DD MS/MS is acquired at ultra high resolution for enhanced signal to noise. Please note that at the higher resolution, the signal to noise is exceptional thus allowing much lower limits of detection than demonstrated.
136.11 136.12 136.13m/z
0
20
40 136.1206
136.1246
150.10 150.12 150.14m/z
0
20
40
150.0999
150.1363
244.20m/z
0
20
40244.1914
244.2471
300.15 300.20m/z
0
20
40
300.1870
304.15 304.20m/z
0
20
40
304.1763304.1373
318.1 318.2m/z
0
20
40 318.2284
318.1919100
n
278.15 278.20m/z
0
50278.2180g
Conclusion• We have shown an easy to use technique (no sample preparation, no chromatography)
that shows extraordinary potential for the semi-quantitative screening of drugs of abuse in forensic toxicology.
identification, allow for unlimited number of analytes in the method and retrospective data analysis is possible because a full MS spectrum is recorded in addition to All Ion Fragmentation (AIF) or Data Dependent (DD) MS/MS.
In this work, the ability of paper spray coupled to a very sensitive and fast Orbitrapanalyzer is explored for its potential as a forensic toxicology screening tool
demonstrated.
Quantitation• Amitriptyline-spiked in blood (10–5,000 ng/mL) yielded limits of quantitation (LOQ) of
25 ng/mL using amitriptyline-d3 as internal standard (Figure 6).
FIGURE 5. DD MS/MS fragmentation at the highest resolving power of 140,000 (FWHM at m/z 200) in the Q Exactive allows for sensitive detection. An enhanced signal to noise ratio (as compared to MS/MS at 17,500 resolving power, data not shown) is observed. Accurate mass on both precursor and fragments (4-5 ppmand 1-3 ppm, respectively) are used for the identification of compounds in
• Any combination of user required experiments, e.g., MS, AIF and Data Dependent MS/MS, are allowed for the best hit confirmation in a single experiment.
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern
analyzer is explored for its potential as a forensic toxicology screening tool.
MethodsSample Preparation
Mixtures of drugs (Cerilliant TX) were spiked in blood (bovine blood Lampire
FIGURE 2. Full scan MS experiments - highest resolving powers, e.g., 70,000 and 140,000 (FWHM at m/z 200), are required for the identification of drugs from DBS due to matrix interference. Bovine blood spiked with six drugs, four drugs shown below Resolving power from 17 500 35 000 70 000 and 140 000 top to bottom
• Variability in terms of %RSD (Std Dev/Mean*100) is between <1 to 16% for drug in blood. Figure 6 displays amitriptyline data for dried blood spots.
FIGURE 3. TraceFinder 3.0 software results shown below. Data processed in targeted screening analysis mode. All analytes in the mix are positively id tifi d b t / l d fi d b i t i tt d th
screening applications. Sample: mixture of six drugs analyzed from DBS, four shown below. Concentrations noted in each panel.
a) Amphetamine b) Methamphetamine
20
60
100 91.0544
119.0859
150 1280
NL: 1.78E7
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern matching are required to confirm drugs identified solely by accurate mass (Fig. 3).
• We have demonstrated feasibility for rapid blood analysis for intoxication cases where expected concentrations are high (≥100 ng/mL, Fig. 4). DD MS2 data (Fig. 5) indicates
• Mixtures of drugs (Cerilliant, TX) were spiked in blood (bovine blood, LampireBiologicals, New Jersey) stabilized with K2-EDTA. Blood sample integrity maintained by not exceeding 5% of solvent in blood (v/v).
• Twelve microliters of spiked blood sample were loaded to paper cartridges, dried under a nitrogen gas flow for 20 min and loaded into stackers that hold up to 40 cassettes.
below. Resolving power from 17,500, 35,000, 70,000 and 140,000 top to bottom. The [M+H]+ ion is highlighted by a red line. Mass accuracies 1-2 ppm.
identified by exact m/z values and confirmed by isotopic pattern and the presence of two fragments from the AIF experiment (see Table).
Data collected with the Exactive Plus mass spectrometer.60
100 91.0544
119.0858 136 0736
NL: 5.86E61000 ng/mL 1000 ng/mL
a) Amphetamine b) Methamphetamine
20
60
100
20 150.1280
91.0544
119.0859
150.1280
NL: 9.06E6
p g ( g , g ) ( g )lower levels can be achieved and this is part of ongoing investigations.
• High resolution and accurate mass are crucial techniques for analyzing complex samples by MS and nicely complement the paper spray technique in the screening of drugs from d i d bl d t
• Solvent is automatically dispensed to the DBS before analysis and an applied high voltage (3-5 kV) induces electrospray from the sharp tip of the paper (Figure 1).
• The extraction solvent used in this work is 95/5 (v/v) methanol/water with 100 ppm acetic acid (pH 4.5).
244.2284244.2069
300.1592
300.2911
318.1711318.1964
318 13916080
100 136.0738
amphetamine PCP codeine cocaethylene
17 500
RP20
60
100
20119.0858 136.0736
91.0544
136.0736119.0858
NL: 2.68E6
500 ng/mL 500 ng/mL
100 120 1400
20
60
100 149.023791.0544
119.0858
NL: 1.59E6
dried blood spots.
• Data collected in this screening application allows for retrospective analysis as a full scan MS event is always acquired.
Mass Spectrometry• The paper-spray source was coupled to either a Thermo Scientific™ Exactive Plus™ or
a Thermo Scientific™ Q Exactive™ Orbitrap mass spectrometer. • An automated experiment for drug screening consisted of 30 sec data collection,
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and
244.2071
244.2269
300.2190
300.1614
300.1810300.2907
300 2186
318.1391
318.2667318.1716
318.1916
406080
100
tive
Abu
ndan
ce 02040 136.1130
136.0738136.1127
136.1055
17,500
35,00090 100 110 120 130
020
60
100 136.0736
91.0543
119.0857
NL: 1.18E6100 ng/mL 100 ng/mL
60
100 196.1336
82 0653
NL: 6.90E7
100 120 140
60
100 86.0966
159.1173
91 0545
NL: 4.33E7
• The paper spray technique coupled with automated data processing using TraceFinder3.0 software provides a complete solution for drug screening in forensic toxicology.
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and Data Dependent Higher Collision Dissociation (HCD) MS/MS (Q Exactive MS).
• For maximum specificity and sensitivity, both full scan and fragmentation data were acquired at 140,000 resolving power (FWHM at m/z 200). Normalized collision energy was 40 eV.
244.2068
244.2268
300.2186
300.1606
300.2912300.1794
300.1433
318.1432318.2648
318.1715
318.1414 318 2840318.2286
020406080
1000
20
Rel
at
136.1127
136.0739
136.105770,000
Isotopic pattern match
Simulation
90 100 110 120 130
c) PCP d) Cocaethylene
References1000 ng/mL 1000 ng/mL
20
60
100
2082.0653
150.0917 318.1704196.1336
82.0653150 0917
NL: 3.74E7
20
60
100
2091.0545
244.206486.0966
159.1172
91.0544
NL: 2.66E7
• All data acquisition used the Thermo Scientific™ Xcalibur™ sequences and contact closure trigger from the paper spray source.
Data Analysis• Thermo Scientific™ QualBrowser™ software from the Xcalibur platform was used for 244.15 244.20 244.25
244.2066
244.2269
300.1 300.2 300.3
300.1608
300.2910300.2185300.1431
300.3274
318.1 318.2 318.3
318.2840318.1711
318.1417318.1907318.2836136.10
020406080
1000 136.1127
136.0739
136.1056136.0845
140,000
ExperimentalReferences1. Manicke, N.; Yang, Q.; Wang, H.; Oradu, S.; Ouyang, Z.; Cooks, R.G. Assessment of
Paper Spray Ionization for Quantitation of Pharmaceuticals in Blood Spots. IJMS 2011, 300, 123-129.500 ng/mL 500 ng/mL
020
60
100
20 150.0917 318.1705196.1336
80.9480
150.0917105.0337
318.1704
NL: 6.60E6
020
60
100
20 104.1071 244.206386.0966
245.1365159.1172
95.0857135.1171
213.1100
NL: 3.26E6
Thermo Scientific QualBrowser software from the Xcalibur platform was used for spectra visualization. TraceFinder 3.0 software was used for the automated identification and confirmation in the targeted screening of drugs.
For forensic toxicology use only.
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others
m/z m/z m/zm/z
100 ng/mL 100 ng/mL
50 100 150 200 250 3000
100 150 200 2500 intellectual property rights of others.
PO64316-EN 1114S
6 Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic Toxicology
Direct Analysis using Paper-Spray Mass Spectrometry: Method Development for the Rapid Screening of Drugs of Abuse for Forensic ToxicologyMaria C. Prieto Conaway1, Nicholas E. Manicke2, Marta Kozak1
1Thermo Fisher Scientific 355 River Oaks Parkway San Jose CA 95134 2Department of Chemistry Indiana University Purdue University Indianapolis IN1Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose CA 95134, 2Department of Chemistry, Indiana University-Purdue University Indianapolis, IN
Overview FIGURE 6b. Quantitative results for it i t li li d b i t l
FIGURE 6a. Full scan MS spectra for
Purpose: Method development for the rapid and semi-quantitative screening of drugs of abuse in forensic toxicology using paper spray mass spectrometry.
Methods: Bovine blood spiked with common drugs of abuse and analyzed as dried blood spots by paper spray ionization/Orbitrap mass spectrometry. High resolution and accurate
FIGURE 1. Prototype paper spray ion source (Prosolia, Inc., IN) showing, clockwise from top left: paper spray ion source, mechanism for dispensing solvent to the sample, paper cassette indicating sample deposition and DBS-spotted paper cassette electrospraying into mass spectrometer inlet.
ResultsScreening for drugs of abuse: resolving power, accurate mass for compound identification• Figure 2 shows that high and ultrahigh resolving powers (70,000 and 140,000 FWHM
FIGURE 4. Accurate mass (3-4 ppm) MS spectra at 140,000 resolving power (FWHM at m/z 200) showing drugs detected down to 100 ng/mL. Sample contained six drugs analyzed from DBS.
meth-
amitriptyline normalized by internal standard from DBS samples. Calibration curve and %RSD variability (n=3) shown.
the [M+H]+ ion of amitriptyline at various concentrations from DBS samples. Acquired at 70,000 resolving power.
Mass accuracy 2-3 ppmAmitriptyline
Y = -0.0519284+0.001756*X R^2 = 0.9933 W: 1/X
9p y p p p y p p y gmass used in full MS, MS2 and All Ion Fragmentation experiments for the identification and confirmation of drugs from dried blood spot samples. Thermo Scientific™ TraceFinder™ 3.0 software used for data analysis.
Results: Able to identify six drugs of abuse from dried blood spots at a 100 ng/mL level with outstanding signal to noise Limit of detection from dried blood spots with this
g g g g p ( , ,at m/z 200) are required when evaluating samples from complex matrices with no sample preparation and no prior chromatographic separation. Mass accuracies 1-2 ppm at the higher resolving powers (70,000 and 140,000, FWHM at m/z 200).
• Results from TraceFinder software, which is effectively used for targeted or unknown screening analysis, are neatly summarized in Figure 3. All six drugs are positively 40
60
80
100136.1125
40
60
80
100150.1282
150.1313 40
60
80
100244.2067
40
60
80
100300.1604
300.1661
300.1787300.142640
60
80
100304.1552
40
60
80
100318.1710
0 ng
/mL
amphetamine PCP codeine cocaethylenecocainemeth-amphetamine
50
1000
50
100278.1911
278.1910
5000 ng/mL
1000 ng/mL
Mass accuracy 2 3 ppm
3
4
5
6
7
8
9
Are
a R
atio
with outstanding signal to noise. Limit of detection from dried blood spots with this technique is 1-10 ng/mL, compound dependent. Paper spray is easy to use, requires no sample preparation and no prior chromatography, making for a quick technique with the potential to identify compounds in seconds. The Thermo Scientific™ Orbitrap™ Exactive™ family of mass spectrometers are ideally suited for coupling to paper spray ionization.
g y , y g g p yidentified from a dry blood spot sample.
Screening for drugs of abuse at various concentrations• A drug mixture of six compounds was analyzed at 100, 500, 1000 and 2500 ng/mL for
forensic toxicology screening Amphetamine methamphetamine cocaine60
80
1000
20
40
136.1222
136.1125
60
80
100
bund
ance
0
20
40
150.1212
150.1313
150.1282
60
80
100
bund
ance
0
20
40
244.2277
244.1916 244.2475244.2067
244.227860
80
100
bund
ance
0
20
40
300.2024
300.1427
300.1604300.1661
300.1790 60
80
100
bund
ance
0
20
40
304.1910304.1458
304.1646
304.1552
60
80
100
bund
ance
0
20
40
318.1918318.1405
318.2283
318.1708
2500
0 ng
/mL
1000
50
1000
50
278.1910
278.1909
g
500 ng/mL
250 ng/mL
0 1000 2000 3000 4000 5000ng/mL
0
1
2
IntroductionPaper spray is a direct ionization technique that simplifies the mass spectrometric analysis of dried blood spots (DBS). Paper-spray technology is therefore attractive for forensic toxicology screening for drugs of abuse. The sample collection and storage of DBS in a
f f f
forensic toxicology screening. Amphetamine, methamphetamine, cocaine, cocaethylene, codeine and PCP are shown in this work.
• This group of samples were detected by full scan MS down to 100 ng/mL levels (Figure 4) (140,000 resolving power; FWHM at m/z 200).
60
80
1000
20
40
136.1222
136.1247
136.1125
60
80
1000
20
40
Rel
ativ
e A
b
150.1211
150.1282
60
80
1000
20
40
Rel
ativ
e A
b
244.1915 244.2470
244.2067244.2278
60
80
1000
20
40
Rel
ativ
e A
b
300.2024
300.1426
300.1787
300.1603
60
80
1000
20
40
Rel
ativ
e A
b
304.1973
304.1760
304.1552
60
80
1000
20
40
Rel
ativ
e A
b
318.1403318.1919
318.1708
1000
ng/m
L
1000
50
1000
50
278.1910
278.2183
278.1909
250 ng/mL
100 ng/mL
50 ng/mL
Level (ng/mL) % RSD
simple paper cassette make shipment of samples to the forensic toxicology lab safe and convenient. Both qualitative and quantitative analysis of small molecules from complex matrices such as blood or other biological fluids is possible without time consuming sample preparation and chromatography.
Quantitation of DBS samples with paper spray MS is fairly well established even though a
Fragmentation and isotopic pattern matching for compound confirmation• Accurate mass m/z values were used for identification of screened drugs. Isotopic
pattern matching and two fragments from the AIF experiment were used for drug confirmation (TraceFinder table Fig. 3). Alternatively, DD MS/MS from a Q Exactivemass spectrometer can be used. 60
80
1000
20
40
136.1222
136.1247
136.1154
136.1125
60
80
1000
20
40
150.1211
150.1130150.0996
150.1211
150.1130
150.128260
80
1000
20
40
244.1914244.2471
244.2278
244.2066
60
80
1000
20
40
300.2024
300.1426
300.1661 60
80
1000
20
40
304.1891304.1377304.1551
60
80
1000
20
40
318.1404
318.1919
318.1708318.1402
500
ng/m
L
1000
50
1000
50 278.2180
278.1911
278.2182
278.1909278 2180
25 ng/mL
10 ng/mL300.1603
Conclusion
Quantitation of DBS samples with paper-spray MS is fairly well established even though a commercial product is not yet available (1). While previous work used a Thermo Scientific triple quadrupole mass spectrometer and monitored specific MS/MS transitions, full-MS instruments with Orbitrap analyzers are ideally suited as rapid screening tools. Orbitrapanalyzers provide high resolution, accurate mass (HR/AM) analysis for high confidence identification allow for unlimited number of analytes in the method and retrospective data
p• Figure 5 shows accurate mass fragmentation spectra by targeted DD MS/MS for a
DBS sample containing a mixture of 6 drugs. DD MS/MS is acquired at ultra high resolution for enhanced signal to noise. Please note that at the higher resolution, the signal to noise is exceptional thus allowing much lower limits of detection than demonstrated.
136.11 136.12 136.13m/z
0
20
40 136.1206
136.1246
150.10 150.12 150.14m/z
0
20
40
150.0999
150.1363
244.20m/z
0
20
40244.1914
244.2471
300.15 300.20m/z
0
20
40
300.1870
304.15 304.20m/z
0
20
40
304.1763304.1373
318.1 318.2m/z
0
20
40 318.2284
318.1919100
n
278.15 278.20m/z
0
50278.2180g
Conclusion• We have shown an easy to use technique (no sample preparation, no chromatography)
that shows extraordinary potential for the semi-quantitative screening of drugs of abuse in forensic toxicology.
identification, allow for unlimited number of analytes in the method and retrospective data analysis is possible because a full MS spectrum is recorded in addition to All Ion Fragmentation (AIF) or Data Dependent (DD) MS/MS.
In this work, the ability of paper spray coupled to a very sensitive and fast Orbitrapanalyzer is explored for its potential as a forensic toxicology screening tool
demonstrated.
Quantitation• Amitriptyline-spiked in blood (10–5,000 ng/mL) yielded limits of quantitation (LOQ) of
25 ng/mL using amitriptyline-d3 as internal standard (Figure 6).
FIGURE 5. DD MS/MS fragmentation at the highest resolving power of 140,000 (FWHM at m/z 200) in the Q Exactive allows for sensitive detection. An enhanced signal to noise ratio (as compared to MS/MS at 17,500 resolving power, data not shown) is observed. Accurate mass on both precursor and fragments (4-5 ppmand 1-3 ppm, respectively) are used for the identification of compounds in
• Any combination of user required experiments, e.g., MS, AIF and Data Dependent MS/MS, are allowed for the best hit confirmation in a single experiment.
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern
analyzer is explored for its potential as a forensic toxicology screening tool.
MethodsSample Preparation
Mixtures of drugs (Cerilliant TX) were spiked in blood (bovine blood Lampire
FIGURE 2. Full scan MS experiments - highest resolving powers, e.g., 70,000 and 140,000 (FWHM at m/z 200), are required for the identification of drugs from DBS due to matrix interference. Bovine blood spiked with six drugs, four drugs shown below Resolving power from 17 500 35 000 70 000 and 140 000 top to bottom
• Variability in terms of %RSD (Std Dev/Mean*100) is between <1 to 16% for drug in blood. Figure 6 displays amitriptyline data for dried blood spots.
FIGURE 3. TraceFinder 3.0 software results shown below. Data processed in targeted screening analysis mode. All analytes in the mix are positively id tifi d b t / l d fi d b i t i tt d th
screening applications. Sample: mixture of six drugs analyzed from DBS, four shown below. Concentrations noted in each panel.
a) Amphetamine b) Methamphetamine
20
60
100 91.0544
119.0859
150 1280
NL: 1.78E7
• Accurate mass fragments (from AIF or DD MS/MS experiment) and isotopic pattern matching are required to confirm drugs identified solely by accurate mass (Fig. 3).
• We have demonstrated feasibility for rapid blood analysis for intoxication cases where expected concentrations are high (≥100 ng/mL, Fig. 4). DD MS2 data (Fig. 5) indicates
• Mixtures of drugs (Cerilliant, TX) were spiked in blood (bovine blood, LampireBiologicals, New Jersey) stabilized with K2-EDTA. Blood sample integrity maintained by not exceeding 5% of solvent in blood (v/v).
• Twelve microliters of spiked blood sample were loaded to paper cartridges, dried under a nitrogen gas flow for 20 min and loaded into stackers that hold up to 40 cassettes.
below. Resolving power from 17,500, 35,000, 70,000 and 140,000 top to bottom. The [M+H]+ ion is highlighted by a red line. Mass accuracies 1-2 ppm.
identified by exact m/z values and confirmed by isotopic pattern and the presence of two fragments from the AIF experiment (see Table).
Data collected with the Exactive Plus mass spectrometer.60
100 91.0544
119.0858 136 0736
NL: 5.86E61000 ng/mL 1000 ng/mL
a) Amphetamine b) Methamphetamine
20
60
100
20 150.1280
91.0544
119.0859
150.1280
NL: 9.06E6
p g ( g , g ) ( g )lower levels can be achieved and this is part of ongoing investigations.
• High resolution and accurate mass are crucial techniques for analyzing complex samples by MS and nicely complement the paper spray technique in the screening of drugs from d i d bl d t
• Solvent is automatically dispensed to the DBS before analysis and an applied high voltage (3-5 kV) induces electrospray from the sharp tip of the paper (Figure 1).
• The extraction solvent used in this work is 95/5 (v/v) methanol/water with 100 ppm acetic acid (pH 4.5).
244.2284244.2069
300.1592
300.2911
318.1711318.1964
318 13916080
100 136.0738
amphetamine PCP codeine cocaethylene
17 500
RP20
60
100
20119.0858 136.0736
91.0544
136.0736119.0858
NL: 2.68E6
500 ng/mL 500 ng/mL
100 120 1400
20
60
100 149.023791.0544
119.0858
NL: 1.59E6
dried blood spots.
• Data collected in this screening application allows for retrospective analysis as a full scan MS event is always acquired.
Mass Spectrometry• The paper-spray source was coupled to either a Thermo Scientific™ Exactive Plus™ or
a Thermo Scientific™ Q Exactive™ Orbitrap mass spectrometer. • An automated experiment for drug screening consisted of 30 sec data collection,
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and
244.2071
244.2269
300.2190
300.1614
300.1810300.2907
300 2186
318.1391
318.2667318.1716
318.1916
406080
100
tive
Abu
ndan
ce 02040 136.1130
136.0738136.1127
136.1055
17,500
35,00090 100 110 120 130
020
60
100 136.0736
91.0543
119.0857
NL: 1.18E6100 ng/mL 100 ng/mL
60
100 196.1336
82 0653
NL: 6.90E7
100 120 140
60
100 86.0966
159.1173
91 0545
NL: 4.33E7
• The paper spray technique coupled with automated data processing using TraceFinder3.0 software provides a complete solution for drug screening in forensic toxicology.
switching between full scan and AIF experiments (Exactive Plus MS) or full scan and Data Dependent Higher Collision Dissociation (HCD) MS/MS (Q Exactive MS).
• For maximum specificity and sensitivity, both full scan and fragmentation data were acquired at 140,000 resolving power (FWHM at m/z 200). Normalized collision energy was 40 eV.
244.2068
244.2268
300.2186
300.1606
300.2912300.1794
300.1433
318.1432318.2648
318.1715
318.1414 318 2840318.2286
020406080
1000
20
Rel
at
136.1127
136.0739
136.105770,000
Isotopic pattern match
Simulation
90 100 110 120 130
c) PCP d) Cocaethylene
References1000 ng/mL 1000 ng/mL
20
60
100
2082.0653
150.0917 318.1704196.1336
82.0653150 0917
NL: 3.74E7
20
60
100
2091.0545
244.206486.0966
159.1172
91.0544
NL: 2.66E7
• All data acquisition used the Thermo Scientific™ Xcalibur™ sequences and contact closure trigger from the paper spray source.
Data Analysis• Thermo Scientific™ QualBrowser™ software from the Xcalibur platform was used for 244.15 244.20 244.25
244.2066
244.2269
300.1 300.2 300.3
300.1608
300.2910300.2185300.1431
300.3274
318.1 318.2 318.3
318.2840318.1711
318.1417318.1907318.2836136.10
020406080
1000 136.1127
136.0739
136.1056136.0845
140,000
ExperimentalReferences1. Manicke, N.; Yang, Q.; Wang, H.; Oradu, S.; Ouyang, Z.; Cooks, R.G. Assessment of
Paper Spray Ionization for Quantitation of Pharmaceuticals in Blood Spots. IJMS 2011, 300, 123-129.500 ng/mL 500 ng/mL
020
60
100
20 150.0917 318.1705196.1336
80.9480
150.0917105.0337
318.1704
NL: 6.60E6
020
60
100
20 104.1071 244.206386.0966
245.1365159.1172
95.0857135.1171
213.1100
NL: 3.26E6
Thermo Scientific QualBrowser software from the Xcalibur platform was used for spectra visualization. TraceFinder 3.0 software was used for the automated identification and confirmation in the targeted screening of drugs.
For forensic toxicology use only.
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This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others
m/z m/z m/zm/z
100 ng/mL 100 ng/mL
50 100 150 200 250 3000
100 150 200 2500 intellectual property rights of others.
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