A Generic LC-MS Method for the Analysis of
Multiple of Drug of Abuse Classes with the Thermo
Scientific ExactiveTM System
Kent Johnson
Fortes lab, Wilsonville Oregon
Forensic Toxicology Use Only
List of drug of abuse candidates for LC-MS analysis
Sample matrix: urine and blood
Benzodiazepines
7-Aminonitrazepam
7-Aminoclonazepam
7-Aminoflunitrazepam
2-Hydroxy-ethyl-flurazepam
Desalkylflurazepam
Diazepam
Hydroxy-alprazolam
Hydroxy-triazolam
Nordiazepam
Lorazepam
Oxazepam
Temazepam
Opiates
Morphine
Hydromorphone
Oxymorphone
Codeine
Dihydrocodeine
Hydrocodone
Oxycodone
Meperidine
Normeperidine
Other drugs group 1
Ketamine
Norketamine
Butorphanol
Fentanyl
Norfentanyl
Nalbuphine
Alfentanil
Sulfentanil
Zolpidem
Trazodone
Venlafaxine
Zopiclone
Forensic Toxicology Use Only
2
Other drugs group 2
Methylphenidate
Ritalinic Acid
Dextromethorphan
Dextrophan
Propoxyphene
Norpropoxyphene
6-MAM
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
Forensic Toxicology Use Only
3
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
Forensic Toxicology Use Only
4
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
10 ng/mL
Benzodiazepines
10 ng/mL
6-MAM
3 ng/mL
Other drugs of
abuse group1
Other drugs of abuse
group 2
2-50 ng/mL
10 ng/mL
Forensic Toxicology Use Only
5
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
Forensic Toxicology Use Only
6
Power of mass resolution – Ethinyl Estradiol
Resolution: 10k, 30k, 50k, 100k
Butyl-Phthalate, 279.15909
(ubiquitous background ion)
100
90
80
Relative Abundance
70
60
Ethinyl-Estradiol, 279.17434
50
40
30
20
10
0
279.12
279.14
m/z
279.16
279.18
Forensic Toxicology Use Only
7
279.20
Exactive LC/MS setup
Forensic Toxicology Use Only
8
Exactive LC/MS setup
Forensic Toxicology Use Only
9
Exactive quantitative analysis workflow
Sample Preparation
SPE or urine dilution
LC-MS method
PFP column, HESI source, Mass resolution 100,000
Data processing
extract chromatograms with 2 ppm mass accuracy
Custom reports
Forensic Toxicology Use Only
10
Urine sample preparation
Benzodiazepines, opiates and
Other drugs of abuse group 2
Other drugs of abuse
group 1
Urine dilution
SPE
Int. std addition
Enzymatic Hydrolysis
SPE Extraction
Evaporation
Reconstitution
Int. std addition
20 X dilution with 25-50% MeOH
Inject Samples
Forensic Toxicology Use Only
11
Blood sample preparation
SPE
Other drugs of abuse
group 1
Ritalinic Acid
Precipitation/dilution
Int. std addition
Int. std addition
SPE Extraction
ACN addition in ratio 1:1
Benzodiazepines, opiates
other drugs of abuse group 2
Evaporation
Centrifugation
Reconstitution
30 X dilution
Inject Sample
Forensic Toxicology Use Only
12
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
Forensic Toxicology Use Only
13
MS method
•
•
•
•
HESI source
Full scan data in compounds specific m/z range
Resolution 100K
Extract chromatograms using 2 ppm mass window
Forensic Toxicology Use Only
14
Opiates and Opioids in Urine
Hydrocodone
Codeine
Morphine
Oxymorphone D3
Hydromorphone
Oxycodone D6
Normeperidine
Dihydrocodeine D6
Oxycodone
Meperidine D6
Oxymorphine
Hydrocodone D6
Codeine D6
dihydrocodeine
Meperidine
Morphine D6
Forensic Toxicology Use Only
15
Hydromorphone D6
Opiates and Opioids in Urine - dynamic range
Linearity range better than 10 – 10,000 ng/mL
Hydrocodone
Y = 0.0744648+0.000852152*X R^2 = 0.9401 W: 1/X^2
11.0
Codeine
Y = 0.0702431+0.000851922*X R^2 = 0.9461 W: 1/X^2
10.5
10.0
10.5
9.5
10.0
Hydrocodone
9.0
8.5
8.0
9.0
8.5
8.0
7.0
7.5
6.5
7.0
6.0
6.5
5.5
Area Ratio
Area Ratio
Codeine
9.5
7.5
5.0
4.5
6.0
5.5
5.0
4.0
4.5
3.5
4.0
3.0
3.5
2.5
3.0
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0
2000
4000
6000
ng/mL
8000
10000
12000
0.0
0
2000
4000
Meperidine
Y = 0.075201+0.000849812*X R^2 = 0.9540 W: 1/X^2
11.5
9.0
10000
Morphine
11.0
Meperidine
9.5
8000
Morphine
Y = 0.054877+0.000912426*X R^2 = 0.9288 W: 1/X^2
10.5
10.0
6000
ng/mL
10.5
10.0
9.5
8.5
9.0
8.0
8.5
8.0
7.0
7.5
6.5
7.0
Area Ratio
Area Ratio
7.5
6.0
5.5
5.0
6.5
6.0
5.5
5.0
4.5
4.5
4.0
4.0
3.5
3.5
3.0
3.0
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0
2000
4000
6000
ng/mL
8000
10000
0
2000
Forensic Toxicology Use Only
16
4000
6000
ng/mL
8000
10000
Opiates and Opioids in Urine- dynamic range
Linearity range 10 – 10,000 ng/mL
Oxymorphone
Y = 0.0572347+0.000794734*X R^2 = 0.9411 W: 1/X^2
Normeperidine
Y = 0.0229096+0.00023572*X R^2 = 0.9434 W: 1/X^2
10.0
9.5
3.0
2.6
Oxymorphone
9.0
Normeperidine
2.8
8.5
8.0
2.4
7.5
2.2
7.0
6.5
2.0
6.0
Area Ratio
Area Ratio
1.8
1.6
1.4
5.5
5.0
4.5
4.0
1.2
3.5
1.0
3.0
0.8
2.5
2.0
0.6
1.5
0.4
1.0
0.5
0.2
0.0
0.0
0
2000
4000
6000
ng/mL
8000
0
10000
10.0
10.0
6000
ng/mL
8000
10000
Dihydrocodeine
9.5
Oxycodone
10.5
4000
Oxymorphone
Y = 0.0572347+0.000794734*X R^2 = 0.9411 W: 1/X^2
Oxycodone
Y= 0.0712176+0.000849364*X R^2= 0.9510 W: 1/X^2
11.0
2000
9.0
8.5
9.5
8.0
9.0
7.5
8.5
8.0
7.0
7.5
6.5
7.0
6.0
Area Ratio
Area Ratio
6.5
6.0
5.5
5.0
5.5
5.0
4.5
4.0
4.5
3.5
4.0
3.0
3.5
3.0
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0
0
2000
4000
6000
ng/mL
8000
2000
10000
Forensic Toxicology Use Only
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4000
6000
ng/mL
8000
10000
GC-MS correlation
No
Analyte
1
2
3
3
4
4
4
5
5
6
7
8
8
8
8
Morphine
Oxymorphone
Oxycodone
Oxymorphone
Hydrocodone
Hydromorphone
Dihydrocodeine
Oxycodone
Oxymorphone
Opiates
Opiates
Morphine
Hydrocodone
Hydromorphone
Dihydrocodeine
Exactive LC-MS GC-MS
(ng/mL)
(ng/mL)
289
343
117
219
167
166
121
174
651
650
503
552
57
74
1833
1822
593
574
Only
0
0
after dilution
0
0
22,562
37,954***
6061
7336
3492
2706
1084
1104
Forensic Toxicology Use Only
18
6-MAM linearity and dynamic range
6AMSTD10 - m/z= 328.15-328.15 SM: 5 RT: 2.10 - 4.10 NL: 3.16E5
F: {0,0} + p ESI Full ms [ 327.00-335.00]
RT: 3.10
100
95
90
85
6-MAM 10 ng/mL
80
75
70
6- MAM 3 - 400 ng/mL
Relative Intensity
65
6-ACETYLMORPHINE
Y = -0.0899199+0.0613337*X R^2 = 0.9984 W: 1/X
26
60
55
50
45
40
35
30
25
20
24
15
10
5
22
3.40
3.75
0
2.2
2.4
2.6
2.8
3.0
3.2
Time (min)
3.4
3.6
3.8
4.0
20
Area Ratio
18
6AMSTD10 - m/z= 334.19-334.19 SM: 5 RT: 2.10 - 4.10 NL: 5.70E5
F: {0,0} + p ESI Full ms [ 327.00-335.00]
16
RT: 3.10
100
14
95
90
12
IS 20 ng/mL
85
80
75
10
70
65
Relative Intensity
8
6
4
60
55
50
45
40
35
30
2
25
20
0
15
0
50
100
150
200
250
NG/ML
300
350
400
10
5
3.41
3.77
0
2.2
2.4
Forensic Toxicology Use Only
19
2.6
2.8
3.0
3.2
Time (min)
3.4
3.6
3.8
3.91
4.04
4.0
6-MAM NLCP compliance test
Measured =10.6 ng/mL
STD10 W50000MOR - m/z= 328.15-328.15 SM: 5 RT: 2.11 - 4.11 NL: 4.31E5
F: {0,0} + p ESI Full ms [ 327.00-335.00]
RT: 3.11
100
95
90
10 ng/mL of 6-MAM
in the presence of 50,000 ng/mL
Morphine
85
80
75
70
Relative Intensity
65
60
55
50
45
40
35
30
25
20
2.86 2.96
2.25
2.35
15
2.81
3.32
3.40
3.48 3.57
3.69
3.76 3.87
2.54
10
3.98
5
0
2.2
2.4
2.6
2.8
3.0
3.2
Time (min)
3.4
3.6
3.8
Forensic Toxicology Use Only
20
4.0
6-MAM GC-MS Correlation - Samples
No
1
2
3
3
4
4
Exactive LC-MS
(ng/mL)
16.6
59.9
21.4
18.4
13
7.8
Forensic Toxicology Use Only
21
GC-MS
(ng/mL)
15.4
56.8
20.6
14.6
10.8
6.6
Blood samples XIC with mass accuracy of 2 ppm
Zolpidem 293 ng/mL
Zolpidem-D6
174876bld - m/z= 308.18-308.18 SM: 5 RT: 2.18 - 7.18 NL: 7.41E5
F: FTMS {0,0} + p ESI Full ms [ 223.00-1000.00]
100
RT: 4.68
100
95
95
90
90
85
85
80
80
75
75
70
70
65
65
60
60
Relative Intensity
Relative Intensity
174876bld - m/z= 314.21-314.21 SM: 5 RT: 2.18 - 7.18 NL: 1.97E6
F: FTMS {0,0} + p ESI Full ms [ 223.00-1000.00]
RT: 4.68
55
50
45
40
55
50
45
40
35
35
30
30
25
25
20
20
15
15
10
10
5
5
5.50
0
2.5
3.0
3.5
4.0
4.5
5.0
Time (min)
5.84
5.5
6.63
6.0
6.5
5.41
0
7.0
2.5
3.0
Dextrorphan 111 ng/mL
3.5
4.0
4.5
5.0
Time (min)
RT: 4.38
7.0
RT: 4.38
95
95
90
90
85
85
80
80
75
75
70
70
65
Relative Intensity
65
Relative Intensity
6.5
Dextrorphan-D3
100
60
55
50
45
40
60
55
50
45
40
35
35
5.53
30
30
25
25
20
20
15
15
5.06
10
10
5
4.90
6.03
5 .22
0
2 .0
2.5
3.0
3.5
4.0
4.5
Ti me (m in)
5.0
5.5
6.0
5
6.2 7
6.58
6.5
5.54
4.7 7
0
2 .0
2.5
3.0
Forensic Toxicology Use Only
22
6.22
6.0
1763 72 - m/z= 261 .20-26 1.20 S M: 5 RT: 1 .88 - 6.88 NL: 1.9 7E 6
F: FTMS {0,0} + p ES I Full ms [ 232.0 0-1000 .00]
1763 72 - m /z= 258 .19-25 8.19 S M: 5 RT: 1 .88 - 6.88 NL: 1.2 8E 6
F : F TM S {0,0} + p ES I F ull m s [ 232.0 0-1000 .00]
100
5.68
5.5
3.5
4.0
4.5
Time (min)
5.0
5.5
5.80
6.1 9
6.0
6.52
6.5
Blood samples XIC with mass accuracy of 2 ppm
Norpropoxyphene-D5
Norpropoxyphene 168 ng/mL
174876bld - m/z= 331.24-331.24 SM: 5 RT: 3.34 - 5.34 NL: 4.42E5
F: {0,0} + p ESI Full ms [ 320.00-1000.00]
174876bld - m/z= 326.21-326.21 SM: 5 RT: 3.34 - 5.34 NL: 2.18E5
F: {0,0} + p E SI Full ms [ 320.00-1000.00]
RT: 4.34
100
RT: 4.34
100
95
95
90
90
85
85
80
80
75
75
70
70
65
Relative Intensity
Relative Intensity
65
60
55
50
45
40
60
55
50
45
40
35
35
30
30
25
25
4.51
20
20
15
15
10
10
4.45
5
5
4.64
0
3.4
3.6
3.8
4.0
4.2
4.4
Time (min)
4.6
4.79
4.8
4.58
0
5.0
3.4
5.2
3.6
Propoxyphene 162 ng/mL
3.8
4.0
4.2
RT: 4 .3 7
5.0
5.2
RT: 4.36
95
95
90
90
85
85
80
80
75
75
70
70
65
Relative Intensity
65
Relative Intensity
5.07
4.8
Propoxyphene-D5
100
60
55
50
45
40
60
55
50
45
40
35
35
30
30
25
25
20
20
15
15
10
10
5
3 .9 2
0
3.4
3.6
3 .8
4 .81
4 .2 2
4 .0
4 .2
4 .4
Tim e (m in)
4.6
4.8
4 .95
5 .0
5
5.18
5 .2
0
3.41
3.60
3.4
3.6
Forensic Toxicology Use Only
23
4.70
4.6
S td 1000 ng_mlbld - m /z= 351.30-351.30 S M: 5 RT: 3.36 - 5.36 NL: 4.35E6
F : {0,0} + p E SI F ull ms [ 320.00-1000.00]
1 74 87 6b ld - m /z= 34 0.23 -3 40 .2 3 S M : 5 RT: 3 .3 7 - 5 .3 7 NL : 7 .5 9E 5
F: {0 ,0 } + p E S I Full m s [ 32 0.00 -1 00 0.00 ]
10 0
4.4
Time (min)
3.73
3.86
3.8
4.06
4.0
4.71
4.17
4.2
4.4
Time (min)
4.6
4.88
4.8
5.01
5.0
5.15
5.25
5.2
Blood samples XIC with mass accuracy of 2 ppm
Ritalinic Acid 293 ng/mL
154147 - m/z= 220.13-220.13 S M: 5 RT: 1.81 - 4.81 NL: 2.30E6
F: FTMS {0,0} + p ESI Full ms [ 218.00-1000.00]
1541 47 - m /z= 249 .24 -2 49.24 S M: 5 RT: 4.30 - 7 .02 NL: 1.30 E 6
F : F TM S {0,0} + p E S I F ull m s [ 218 .0 0-1 000.00 ]
RT: 3.31
100
PCP-D5 used as Int Std
95
10 0
90
95
85
90
80
85
75
80
75
70
70
65
65
60
Relative Intensity
Relative Intensity
RT: 5.80
55
50
45
40
35
60
55
50
45
40
35
30
30
25
25
20
20
15
15
10
10
5
5
3.71
0
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
Time (min)
3.6
4 .73
0
3.8
4.0
4.2
4.4
4.6
4.8
4.4
4.6
4.9 3
4 .8
Forensic Toxicology Use Only
24
5.0
5.12
5.34
5.2
5 .4
5.6
5.8
Tim e (m in)
6 .0
6.19
6.38
6.2
6.4
6 .5 3
6 .6
6.69
6.84
6.8
7.0
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
Forensic Toxicology Use Only
25
Acknowledgements
• Marta Kozak, Thermo Fisher Scientific
Forensic Toxicology Use Only
26