A Generic LC-MS Method for the Analysis of Multiple of - Orbitrap
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Forensic Toxicology Use Only A Generic LC-MS Method for the Analysis of Multiple of Drug of Abuse Classes with the Thermo Scientific Exactive TM System Kent Johnson Fortes lab, Wilsonville Oregon
Transcript of A Generic LC-MS Method for the Analysis of Multiple of - Orbitrap
Forensic Toxicology Use Only
A Generic LC-MS Method for the Analysis of
Multiple of Drug of Abuse Classes with the Thermo
Scientific ExactiveTM System
Kent JohnsonFortes lab, Wilsonville Oregon
2Forensic Toxicology Use Only
List of drug of abuse candidates for LC-MS analysis
Benzodiazepines Opiates Other drugs group 1 Other drugs group 2
7-Aminonitrazepam
7-Aminoclonazepam
7-Aminoflunitrazepam
2-Hydroxy-ethyl-flurazepam
Desalkylflurazepam
Diazepam
Hydroxy-alprazolam
Hydroxy-triazolam
Nordiazepam
Lorazepam
Oxazepam
Temazepam
Morphine
Hydromorphone
Oxymorphone
Codeine
Dihydrocodeine
Hydrocodone
Oxycodone
Meperidine
Normeperidine
Ketamine
Norketamine
Butorphanol
Fentanyl
Norfentanyl
Nalbuphine
Alfentanil
Sulfentanil
Zolpidem
Trazodone
Venlafaxine
Zopiclone
Methylphenidate
Ritalinic Acid
Dextromethorphan
Dextrophan
Propoxyphene
Norpropoxyphene
6-MAM
Sample matrix: urine and blood
3Forensic Toxicology Use Only
Methods Employed Prior to LC-MS
• Benzodiazepines � GC-MS
• Opiates � GC-MS
• Other drugs of abuse group 1 � ELISA
• Other drugs of abuse group 2 � not analyzed before
4Forensic Toxicology Use Only
Why switch to LC-MS method?
Benefits of replacing GC-MS• Faster � less need for chromatographic separation
• Less sample prep � no derivatization
• No thermal instability � benzodiazepines analysis
• No volatility limitations
Benefits of replacing immunoassay
• Lower consumables cost
• More specific
More cost efficient and analytically more universal
5Forensic Toxicology Use Only
Goal
• Develop fast, easy to use, generic LC-MS method to analyze
multiple classes of drugs of abuse in urine
• Method has to meet industry standards for• Precision
• Accuracy
• Limit of quantitation
• Robustness
• With simplest sample prep procedure
Opiates Benzodiazepines 6-MAM Other drugs of abuse group1
Other drugs of abuse group 2
10 ng/mL 10 ng/mL 3 ng/mL 2-50 ng/mL 10 ng/mL
6Forensic Toxicology Use Only
Exactive uHRAM LC-MS system
• Resolution•100,000 at 1 scan per sec •10,000 at 10 scans per sec
•Mass accuracy•Sub ppm
• Scan speed•Up to 10 scans per second
••Mass range
•m/z 50 - 4000
• Polarity switching
7Forensic Toxicology Use Only
Power of mass resolution – Ethinyl Estradiol
Resolution: 10k, 30k, 50k, 100k
279.12 279.14 279.16 279.18 279.20m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative A
bundance
Ethinyl-Estradiol, 279.17434
Butyl-Phthalate, 279.15909
(ubiquitous background ion)
8Forensic Toxicology Use Only
Exactive LC/MS setup
9Forensic Toxicology Use Only
Exactive LC/MS setup
10Forensic Toxicology Use Only
Exactive quantitative analysis workflow
Sample PreparationSPE or urine dilution
Data processingextract chromatograms with 2 ppm mass accuracy
Custom reports
LC-MS methodPFP column, HESI source, Mass resolution 100,000
11Forensic Toxicology Use Only
Urine sample preparation
SPEInt. std addition
Enzymatic Hydrolysis
SPE Extraction
Evaporation
Reconstitution
Urine dilutionInt. std addition
20 X dilution with 25-50% MeOH
Inject Samples
Benzodiazepines, opiates and
Other drugs of abuse group 2Other drugs of abuse
group 1
12Forensic Toxicology Use Only
Blood sample preparation
SPEInt. std addition
SPE Extraction
Evaporation
Reconstitution
Inject Sample
Benzodiazepines, opiatesother drugs of abuse group 2
Precipitation/dilution
Int. std addition
ACN addition in ratio 1:1
Centrifugation
30 X dilution
Other drugs of abuse group 1
Ritalinic Acid
13Forensic Toxicology Use Only
LC method
• 150 x 2.1 mm, 5 um PFP column
• Mobile phase• A: 20 mm NH4Ac, 0.1% FA in DIW
• B: 0.1% FA in ACN
• 7 min gradient
14Forensic Toxicology Use Only
MS method
• HESI source
• Full scan data in compounds specific m/z range
• Resolution 100K
• Extract chromatograms using 2 ppm mass window
15Forensic Toxicology Use Only
Opiates and Opioids in Urine
Hydrocodone
Codeine
Morphine Hydromorphone
Oxycodone
dihydrocodeine
Normeperidine
Oxymorphine
Meperidine
Oxymorphone D3
Oxycodone D6
Dihydrocodeine D6
Meperidine D6
Hydrocodone D6
Hydromorphone D6
Codeine D6
Morphine D6
16Forensic Toxicology Use Only
HydrocodoneY = 0.0744648+0.000852152*X R^2 = 0.9401 W: 1/X^2
0 2000 4000 6000 8000 10000 12000
ng/mL
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
Are
a R
atio
CodeineY = 0.0702431+0.000851922*X R^2 = 0.9461 W: 1/X^2
0 2000 4000 6000 8000 10000
ng/mL
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
Are
a R
atio
MeperidineY = 0.075201+0.000849812*X R̂ 2 = 0.9540 W: 1/X̂ 2
0 2000 4000 6000 8000 10000
ng/mL
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
Are
a R
atio
MorphineY = 0.054877+0.000912426*X R̂ 2 = 0.9288 W: 1/X̂ 2
0 2000 4000 6000 8000 10000
ng/mL
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
Are
a R
atio
Hydrocodone Codeine
Meperidine Morphine
Opiates and Opioids in Urine - dynamic range
Linearity range better than 10 – 10,000 ng/mL
17Forensic Toxicology Use Only
OxymorphoneY = 0.0572347+0.000794734*X R̂ 2 = 0.9411 W: 1/X̂ 2
0 2000 4000 6000 8000 10000
ng/mL
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
Are
a R
atio
NormeperidineY = 0.0229096+0.00023572*X R̂ 2 = 0.9434 W: 1/X̂ 2
0 2000 4000 6000 8000 10000
ng/mL
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
Are
a R
atio
OxymorphoneY = 0.0572347+0.000794734*X R̂ 2 = 0.9411 W: 1/X̂ 2
0 2000 4000 6000 8000 10000
ng/mL
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
Are
a R
atio
OxycodoneY = 0.0712176+0.000849364*X R̂2 = 0.9510 W: 1/X̂2
0 2000 4000 6000 8000 10000
ng/mL
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
Are
a R
atio
Normeperidine Oxymorphone
Oxycodone Dihydrocodeine
Opiates and Opioids in Urine- dynamic range
Linearity range 10 – 10,000 ng/mL
18Forensic Toxicology Use Only
GC-MS correlation
No AnalyteExactive LC-MS
(ng/mL)
GC-MS
(ng/mL)
1 Morphine 289 343
2 Oxymorphone 117 219
3 Oxycodone 167 166
3 Oxymorphone 121 174
4 Hydrocodone 651 650
4 Hydromorphone 503 552
4 Dihydrocodeine 57 74
5 Oxycodone 1833 1822
5 Oxymorphone 593 574
6 Opiates 0 0
7 Opiates 0 0
8 Morphine 22,562 37,954***
8 Hydrocodone 6061 7336
8 Hydromorphone 3492 2706
8 Dihydrocodeine 1084 1104
Only
after dilution
19Forensic Toxicology Use Only
6AMSTD10 - m/z= 328.15-328.15 SM: 5 RT: 2.10 - 4.10 NL: 3.16E5F: {0,0} + p ESI Full ms [ 327.00-335.00]
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
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80
85
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100
Re
lative
In
ten
sity
RT: 3.10
3.403.75
6-MAM 10 ng/mL
6AMSTD10 - m/z= 334.19-334.19 SM: 5 RT: 2.10 - 4.10 NL: 5.70E5F: {0,0} + p ESI Full ms [ 327.00-335.00]
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
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80
85
90
95
100
Re
lative
In
ten
sity
RT: 3.10
3.913.41 3.77 4.04
IS 20 ng/mL
6-MAM linearity and dynamic range
6- MAM 3 - 400 ng/mL
6-ACETYLMORPHINE
Y = -0.0899199+0.0613337*X R^2 = 0.9984 W: 1/X
0 50 100 150 200 250 300 350 400NG/ML
0
2
4
6
8
10
12
14
16
18
20
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24
26
Are
a R
atio
20Forensic Toxicology Use Only
STD10 W50000MOR - m/z= 328.15-328.15 SM: 5 RT: 2.11 - 4.11 NL: 4.31E5F: {0,0} + p ESI Full ms [ 327.00-335.00]
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
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100
Re
lative In
tensity
RT: 3.11
3.32 3.482.86 3.572.96
3.693.402.25 2.812.35
3.762.54 3.873.98
Measured =10.6 ng/mL
6-MAM NLCP compliance test
10 ng/mL of 6-MAM in the presence of 50,000 ng/mL
Morphine
21Forensic Toxicology Use Only
6-MAM GC-MS Correlation - Samples
NoExactive LC-MS
(ng/mL)
GC-MS
(ng/mL)
1 16.6 15.4
2 59.9 56.8
3 21.4 20.6
3 18.4 14.6
4 13 10.8
4 7.8 6.6
22Forensic Toxicology Use Only
Blood samples XIC with mass accuracy of 2 ppm
174876bld - m/z= 308.18-308.18 SM: 5 RT: 2.18 - 7.18 NL: 7.41E5F: FTMS {0,0} + p ESI Full ms [ 223.00-1000.00]
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Inte
nsity
RT: 4.68
5.845.50 6.63
174876bld - m/z= 314.21-314.21 SM: 5 RT: 2.18 - 7.18 NL: 1.97E6F: FTMS {0,0} + p ESI Full ms [ 223.00-1000.00]
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
Time (min)
0
5
10
15
20
25
30
35
40
45
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55
60
65
70
75
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85
90
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Rela
tive
Inte
nsity
RT: 4.68
5.685.41 6.22
Zolpidem 293 ng/mL Zolpidem-D6
176372 - m /z= 258 .19-258.19 SM: 5 RT: 1 .88 - 6.88 NL: 1.28E6F: FTMS {0,0} + p ES I Full ms [ 232.00-1000 .00]
2 .0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
Time (m in)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative In
ten
sity
RT: 4.38
5.53
6.035 .224.90 6.27 6.58
Dextrorphan 111 ng/mL176372 - m/z= 261.20-261.20 SM: 5 RT: 1.88 - 6.88 NL: 1.97E6F: FTMS {0,0} + p ESI Full ms [ 232.00-1000.00]
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
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100
Re
lative In
tensity
RT: 4.38
5.06
5.54 5.804.77 6.19 6.52
Dextrorphan-D3
23Forensic Toxicology Use Only
Blood samples XIC with mass accuracy of 2 ppm
174876bld - m/z= 326.21-326.21 SM: 5 RT: 3.34 - 5.34 NL: 2.18E5F: {0,0} + p ESI Full ms [ 320.00-1000.00]
3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative In
ten
sity
RT: 4.34
4.51
4.64 4.79
Norpropoxyphene 168 ng/mL174876bld - m/z= 331.24-331.24 SM: 5 RT: 3.34 - 5.34 NL: 4.42E5F: {0,0} + p ESI Full ms [ 320.00-1000.00]
3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive Inte
nsity
RT: 4.34
4.454.58 4.70 5.07
Norpropoxyphene-D5
174876bld - m/z= 340.23-340.23 SM: 5 RT: 3.37 - 5 .37 NL: 7.59E 5F: {0 ,0} + p E SI Full ms [ 320.00-1000.00]
3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
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100
Rela
tive
Inte
nsity
RT: 4 .37
4.954.81 5.183.92 4.22
Propoxyphene 162 ng/mLStd 1000 ng_mlbld - m/z= 351.30-351.30 SM: 5 RT: 3.36 - 5.36 NL: 4.35E6F: {0,0} + p ESI Full ms [ 320.00-1000.00]
3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
In
ten
sity
RT: 4.36
4.71 4.88 5.01 5.15 5.253.86 4.063.41 3.733.60 4.17
Propoxyphene-D5
24Forensic Toxicology Use Only
154147 - m/z= 220.13-220.13 SM: 5 RT: 1.81 - 4.81 NL: 2.30E6F: FTMS {0,0} + p ESI Full ms [ 218.00-1000.00]
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Inte
nsity
RT: 3.31
3.71
Ritalinic Acid 293 ng/mL
154147 - m/z= 249.24-249.24 S M: 5 RT: 4.30 - 7 .02 NL: 1.30E6F: FTMS {0,0} + p ESI Full ms [ 218 .00-1000.00 ]
4.4 4.6 4 .8 5.0 5.2 5 .4 5.6 5.8 6 .0 6.2 6.4 6 .6 6.8 7.0
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
In
ten
sity
RT: 5.80
6.19 6.53 6.844.73 6.38 6.694.93 5.345.12
PCP-D5 used as Int Std
Blood samples XIC with mass accuracy of 2 ppm
25Forensic Toxicology Use Only
Conclusions
• Exactive ultra high resolution MS coupled to LC system• Well suited for quantitative analysis
• Makes addition of new compound to the method easy
• Sensitive and specific LC-MS platform
• Robust for urine dilution protocol for many compounds
• Method can be multiplexed for higher throughput
26Forensic Toxicology Use Only
Acknowledgements
• Marta Kozak, Thermo Fisher Scientific
