A Quantitative Test for Multiple Classes of Illicit ... - Thermo Scientific

Application
Note: 390
Key Words
TSQ Quantum
Discovery MAX
Surveyor HPLC
Forensic drugs
of abuse testing
SRM
A Quantitative Test for Multiple Classes of Illicit
Drugs and Their Primary Metabolites in Human
Biological Fluids by LC-MS/MS for Forensic Use
Kevin J. McHale, 1 Joyce Ho, 2 and Angela Springfield 2
1 Thermo Fisher Scientific, Somerset, NJ, USA; 2 Tarrant County Medical Examiner, Fort Worth, TX, USA
Introduction
Currently, GC/MS is the method of choice for quantifying
drugs of abuse. In recent years, however, many forensic
labs have been switching to LC-MS/MS methods, which
do not require time-consuming derivatization or extensive
sample cleanup necessary in GC/MS analyses. Yet, many
of the LC-MS/MS methods described in the literature
either assay a limited number of illicit drug classes or do
not include their primary metabolites
of these illicit drugs (see table 1). 1-5 Herein is described a
method to assay multiple drugs of abuse including opiates,
stimulants, depressants, and the primary metabolites of
these illicit drugs.
Drug of Abuse
Parent m/z /
Quantifier
Product
m/z
Qualifier
Product
m/z
Ion
Ratio A
Morphine
286
201
165
87
B 7-amino-nitrazepam 252
121
94
14.5
C Ephedrine
166
115
133
95
D Hydromorphone
286
185
157
56
E Amphetamine
136
119
91
86
F
Codeine
300
165
215
97
G 7-amino-clonazepam 286
222
250
85
H Noroxycodone
302
187
227
97
I Methamphetamine 150
91
119
67
J Oxycodone
316
241
256
65
K
MDA
180
135
105
92
L
6-MAM
328
165
211
68
M Norketamine
224
125
179
43
N Hydrocodone
300
199
171
28
O Benzoylecgonine
290
168
105
24
P 7-amino-flunitrazepam 284
135
227
52
Q
MDMA
194
163
135
30
R
Ketamine
238
125
179
40
S
MDEA
208
163
135
32
T Meperidine
248
220
174
55
U Oxazepam
287
241
269
54
V Nordiazepam
271
140
208
82
W
Cocaine
304
182
82
11.1
X Lorazepam
321
275
229
25
Y Nitrazepam
282
236
180
38
Z Alprazolam
309
281
205
85
AA Temazepam
301
255
177
11.8
BB Clonazepam
316
270
214
28
CC Diazepam
285
193
154
70
DD Cocaethylene
318
196
82
15
EE Flunitrazepam
314
268
239
34
FF Methadone
310
265
105
18
Table 1: Summary of SRM transitions for 32 illicit drugs.
Goal
To apply a single LC-MS/MS method to screen for 32
illicit drugs of abuse and their metabolites in biological
fluids.
Experimental Conditions
Sample Preparation
Whole blood or urine samples (0.1–0.4 mL) were spiked
with 20 ng of isotopically labeled internal standards and
purified by solid phase extraction (SPE). Extracted
samples were reconstituted to yield solutions with the
internal standards at 25 ng/mL.
HPLC
HPLC analysis was performed using the Thermo
Scientific Surveyor HPLC System. Each 10 µL sample
was injected directly onto a Thermo Scientific Hypersil
GOLD PFP 50× 2.1 mm, 3 µm analytical column.
A gradient LC method used mobile phases A (0.1%
formic acid in water) and B (0.1% formic acid in
acetonitrile) at a flow rate of 0.3 mL/min.
Mass Spectrometry
MS analysis was carried out on a Thermo Scientific
TSQ Quantum Discovery MAX triple stage quadrupole
mass spectrometer with an electrospray ionization
(ESI) probe. The MS conditions were as follows:
Ion source polarity: Positive ion mode
Ion transfer tube temperature: 370°C
Scan Type: SRM
SRM scan time: 10 ms per transition
Q1, Q3 resolution: unit (0.7 Da FWHM)
Two SRM transitions were monitored for each
component to provide ion ratio confirmations (IRC).
Table 1 summarizes these SRM transitions.

Results and Discussion
Figures 1 and 2 demonstrate the separation of 32 illicit
drugs in less than 10 minutes. Using an SRM dwell time
of 10 ms per transition yielded a minimum of 15 data
points across an LC peak. The limits of quantitation
(LOQs) were determined as either 0.5 ng/mL (lowest calibrator
concentration used) or as the concentration where
the percent relative errors and %CVs were less than 20%
for five replicate injections.
As shown in Figure 3, most calibration curves were
fit using linear regression. Some standards (for example,
cocaine) yielded better statistical calibration curves using
quadratic regression. In these select cases, the target
compound used a structurally different isotopically labeled
internal standard (for example, cocaine used D5-nordiazepam
as internal standard).
The assay of biological sample extracts identified
multiple drugs of abuse and related metabolites. Figures
4A and B demonstrate examples of urine and whole blood
extracts assayed for the presence of illicit drugs with the
RT: 1 . 00
- 5.
00
SM: 5G
Relative Abundance
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
F
AA:
239398
A
AA:
2090313
AA:
782450
LOQ
= 1.
0
G
LOQ
= 2.
5
AA:
925157
LOQ
= 1.
0
H
B
LOQ
= 2.
5
LOQ
= 1.
0
C
LOQ
= 1.
0
D
LOQ
= 1.
0
AA:
2492873
E
AA:
336079
AA:
967868
LOQ
= 1.
0
AA:
381128
1 2 3
Time
( min)
4 5
RT: 2 . 30
- 6.
30
SM: 5G
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
AA:
3732510
I
AA:
896719
J
AA:
950303
LOQ
= 1.
0
LOQ
= 1.
0
K
LOQ
= 1.
0
AA:
955928
L
LOQ
= 2.
5
AA:
1322152
M
LOQ
= 0.
5
AA:
2137498
N
LOQ
= 1.
0
AA:
2257458
O
LOQ
= 0.
5
AA:
1301543
P
LOQ
= 1.
0
3 4 5 6
Time
( min)
developed LC-MS/MS method. Note that cocaine and
benzoylecgonine were detected and qualified below the
assay LOQs in a whole blood extract (Figure 4B), indicating
that lower LOQs are possible for these compounds.
Conclusion
An LC-MS/MS method for assaying illicit drugs and their
metabolites at an LOQ of 0.5–2.5 ng/mL in biological
fluids for forensic use has been demonstrated.
Confirmation of the drugs of abuse was achieved by monitoring
two SRM transitions per compound and measuring
their area ratios to within ±20%. Utilizing a low SRM
dwell time of 10 ms per transition to achieve sufficient
data points across a chromatographic peak had no adverse
effects, such as SRM cross-talk, on the quantitation and
confirmation of these illicit drugs. To authenticate this
assay, extracts from biological fluids were analyzed,
showing the presence of several drugs of abuse and their
metabolites.
RT: 3 . 80
- 7.
80
SM: 5G
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
AA:
5442536
Q
AA:
1338843
T
U
V
W
X
LOQ
= 0.
5
R
LOQ
= 1.
0
AA:
8523737
S
LOQ
= 0.
5
LOQ
= 0.
5
LOQ
= 0.
5
LOQ
= 0.
5
LOQ
= 1.
0
LOQ
= 0.
5
AA:
4459302
AA:
1654085
AA:
642846
AA:
8409524
AA:
668478
4 5 6
Time
( min)
7
Figure 1: Quantifier SRM transitions for the 2.5 ng/mL standard. For the compound designators, refer to the legend in Table 1.
RT: 5 . 80
- 10.
00
SM: 5G
100
50
100
50
100
50
100
50
100
50
0
100
50
100
50
100
50
AA:
925772
AA:
395914
AA:
1770899
AA:
927264
FF
Y
Z
AA
BB
AA:
898565
AA:
9975680
AA:
1618480
LOQ=
0.
5
LOQ
= 1.
0
LOQ
= 1.
0
LOQ
= 1.
0
LOQ
= 1.
0
CC
LOQ=
0.
5
DD
LOQ=
0.
5
EE
LOQ
= 1.
0
AA:
15020389
6 8
Time
( min)
10

RT: 1 . 00
- 5.
00
SM: 5G
Relative Abundance
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
AA:
223730
F
A
Ratio
= 93.
5%
AA:
349752
B
AA:
798147
AA:
581397
Ratio
= 62.
8%
AA:
1943973
Ratio
= 105.
1%
G
Ratio
= 77.
9%
H
AA:
353217
Ratio
= 78.
%0
AA:
753894
Ratio
= 91.
5%
Ratio
= 16.
7%
C
Ratio
= 102.
0%
D
E
AA:
348830
1 2 3
Time
( min)
4 5
RT: 2 . 30
- 6.
30
SM: 5G
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
AA:
2697187
I
AA:
646686
AA:
776874
Ratio
= 72.
3%
J
Ratio
= 72.
1%
K
Ratio
= 81.
8%
AA:
625544
L
Ratio
= 65.
4%
AA:
508634
M
Ratio
= 38.
5%
AA:
704219
N
Ratio
= 32.
9%
AA:
578810
O
AA:
753919
Ratio
= 25.
6%
P
Ratio
= 57.
9%
3 4 5 6
Time
( min)
RT: 3 . 80
- 7.
80
SM: 5G
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
AA:
1667855
Q
Ratio
= 30.
6%
AA:
532928
T
R
Ratio
= 39.
8%
AA:
2759645
S
Ratio
= 56.
6%
U
Ratio
= 57.
2%
V
Ratio
= 76.
5%
W
Ratio
= 11.
4%
X
Ratio
= 32.
4%
Ratio
= 24.
1%
AA:
2525208
AA:
945556
AA:
491609
AA:
957043
AA:
161064
4 5 6
Time
( min)
7
Figure 2: Qualifier SRM transitions for the 2.5 ng/mL standard. For the compound designators and the target ion ratio %, see Table 1.
Area Ratio
Area Ratio
14
13
12
11
10
9
8
7
6
5
4
3
2
1
12
11
10
Morphine
Y = -0.
00536776+
0.
0483892*
X
R^2
=
0.
9977
W:
1/
X
0 20 40 60 80 100 120 140 160 180 200 220 240 260
ng/
mL
9
8
7
6
5
4
3
2
1
Codeine
Y = 0.
00243418+
0.
0417571*
X R^2
= 0.
9960
W:
1/
X
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280
ng/
mL
Area Ratio
Area Ratio
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
Methamphetamine
Y = -0.
0136774+
0.
0507579*
X R^2
= 0.
9938
W:
1/
X
0 20 40 60 80 100 120 140 160 180 200 220 240 260
ng/
mL
MDMA
Y = 0.
0102624+
0.
110954*
X R^2
= 0.
9983
W:
1/
X
0 20 40 60 80 100 120 140 160 180 200 220 240 260
ng/
mL
Area Ratio
Area Ratio
13
12
11
10
9
8
7
6
5
4
3
2
1
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
RT: 5 . 80
- 10.
00
SM: 5G
100
AA:
390127
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
AA:
314805
AA:
171491
AA:
305645
FF
Y
Ratio
= 42.
1%
Z
Ratio
= 79.
5%
AA
Ratio
= 9.
7%
BB
Ratio
= 33.
0%
AA:
575051
CC
AA:
1450888
AA:
505368
Ratio
= 17.
8%
Ratio
= 64.
0%
DD
Ratio
= 14.
5%
EE
Ratio
= 31.
2%
AA:
2673877
6 8
Time
( min)
10
Benzoylecgonine
Y = 0.
0006388+
0.
0465828*
X R^2
= 0.
9994
W:
1/
X
0 20 40 60 80 100 120 140 160 180 200 220 240 260
ng/
mL
Cocaine
Y = -0.
0467479+
0.
718555*
X-0.
000670871*
X^2
R^2
= 0.
9986
W:
1/
X
0 20 40 60 80 100 120 140 160 180 200 220 240 260
ng/
mL
Figure 3: Calibration curves for select drugs of abuse. Regression curve fitting used 1/x weighting from five replicate injections, where R 2 > 0.993 for all standards.

RT: 1 . 00
- 6.
00
SM: 5G
RT:
2.
22
Relative Abundance
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
References
AA:
1407963
D3-Morphine
RT:
2.
22
AA:
50781208
Morphine
Calc.
Conc.
= 786
ng/
mL
RT:
2.
22
AA:
47400472
Morphine
( qualifier)
Ratio
= 93.
3%
1.
84
1 2 3 4 5 6
Time
( min)
1 Kronstrand, R.; Nystrom, I.; Strandberg, J.; Druid, H. “Screening for drugs
of abuse in hair with ion spray LC-MS-MS”; Forensic Sci. Int. 2004,
145(2-3), 183-190.
2 Allen, K.R.; Azad, R.; Field, H.P.; Blake, D.K. “Replacement of immunoassay
by LC tandem mass spectrometry for the routine measurement of drugs
of abuse in oral fluid”; Ann. Clin. Biochem. 2005, 42(4), 277-284.
3 Maralikova, B.; Weinmann, W. “Confirmatory analysis for drugs of abuse
in plasma and urine by high-performance liquid chromatography-tandem
mass spectrometry with respect to criteria for compound identification”;
J. Chromatogr. B. 2004, 811(1), 21-30.
4 Nordgren, H.K.; Beck, O. “Multicomponent screening for drugs of abuse:
direct analysis of urine by LC-MS-MS”; Ther. Drug Monit. 2004, 26(1),
90-97.
5 Edinboro, L.E.; Backer, R.C.; Poklis, A. “Direct analysis of opiates in urine
by liquid chromatography-tandem mass spectrometry”; J Anal Toxicol.
2005, 29(7), 704-710.
View additional Thermo Scientific LC/MS application notes at: www.thermo.com/appnotes
Part of Thermo Fisher Scientific
RT:
3.
61
AA:
3864193
D3-Codeine
3.
05
Codeine
RT:
3.
61
Calc.
Conc.
= 2.
93
ng/
mL
AA:
478862
3.
04
RT:
3.
61
Codeine
( qualifier)
AA:
441541
Ratio
= 92.
2%
RT:
4.
49
AA:
80602828
Benzoylecgonine
Est.
Conc.
= 153
ng/
mL
RT:
4.
48
Benzoylecgonine
( qualifier)
AA:
17676901
Ratio
= 21.
9%
Figure 4A: Assay of urine extract (#423) targeting morphine and its
metabolites. The concentration of benzoylecgonine is estimated because
a labeled internal standard was not added to the sample extract.
RT: 2 . 00
- 8.
00
SM: 7G
Relative Abundance
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
50
RT:
3.
09
AA:
47053
243
Ephedrine
Est.
Conc.
= 209
ng/
mL
RT:
3.
09
AA:
46301
487
Ephedrine
( qualifier)
Ratio
= 98.
4%
RT:
4.
07
AA:
41149894
RT:
4.
08
AA:
2141110
D5-Methamphetamine
Methamphetamine
Calc.
Conc.
= 1.
33
ng/
mL
RT:
4.
08
AA:
1637922
Methamphetamine
( qualifier)
Ratio
= 76.
5%
RT:
4.
48
AA:
176773
Benzoylecgonine
Est.
Conc.
= 0.
19
ng/
mL
RT:
6.
60
AA:
754897
Cocaine
Est.
Conc.
= 0.
22
ng/
mL
RT:
6.
60
Cocaine
( qualifier)
AA:
67552
Ratio
= 8.
9%
2 3 4 5
Time
( min)
6 7 8
Figure 4B: Assay of whole blood extract (#473) targeting amphetamine
and its metabolites. The concentrations of ephedrine, benzoylecgonine
and cocaine are estimated because labeled internal standards were not
added to sample extract.
For Research Use Only. Not for use in diagnostic procedures.
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