Application
Note: 350
Rapid Quantitative and Confirmational
Screening for Drugs in Race Horse Urine
by ESI-LC-MS/MS and MS3
Patrick Russell,1 Paul Steinberg,2 Mary L. Blackburn,2 Diane Cho,2 Robert Heather2
1
University of Florida, Racing Laboratory, Gainesville, Florida; 2Thermo Fisher Scientific, San Jose, California
Key Words
• LTQ™
• Confirmation
• Drug Screening
• MS3
• Quantitation
Introduction
Experimental Conditions
Drugs of abuse in the horse racing industry encompass
a variety of chemical classes and are typically analyzed
from a complex urine matrix. These factors render
the rapid and effective diagnostic screening of these
drugs at low levels difficult. Traditionally, quantitation
has been performed by triple quadrupole mass
spectrometry using reaction monitoring (SRM) mode.
However, this method does not monitor structurally
diagnostic fragmentation. Thus, a second step involving
derivatization and GC/MS confirmation was required.
Sample Preparation
Goal
To develop a simple and fast, yet rugged LC/MS based
method capable of simultaneous qualitative and quantitative analysis. We have evaluated the application of the
Thermo Scientific LTQ linear ion trap mass spectrometer
for providing low levels of detection, good reproducibility,
and wide linear dynamic range required for reliable quantitation, with simultaneous structural confirmation using
diagnostic full-scan MS/MS or MS3 mass spectrometry.
Standards and Unknowns: Standards of the compounds
listed in Table 1 were prepared neat and in urine.
Urine standards and unknowns were spiked, dried,
and reconstituted with 90% water and 10% acetonitrile
with 0.1% acetic acid. Typical Instrument Setup settings
are shown in Figure 1.
HPLC
HPLC System: Thermo Scientific Surveyor™ LC system
Column: Thermo Scientific BETASIL™ C18, 3 µm,
100 × 2.1 mm
Flow Rate: 350 µL/min
Injection Volume: 10 µL (full loop)
Mobile Phase: (A) Water with 0.1% acetic acid
(B) Acetonitrile with 0.1% acetic acid
Gradient: 92% A to 90% B.
MS
Mass Spectrometry: Thermo Scientific LTQ linear ion trap
mass spectrometer
API Source: Thermo Scientific Ion Max™ source with
electrospray ionization (ESI) probe
Ion Transfer Capillary: 220 °C; Sheath Gas: 30 units
Auxiliary Gas: 0 units; Sweep Gas: 20 units
Spray Voltage: 4.5 kV; Isolation Width: 3 amu
Normalized Collision Energy™: 28%
WideBand Activation™: Applied as needed (see Table 1)
Ion Polarity Mode: positive or negative (see Table 1)
Results
Quantitation
Calibration curves were established using neat standards
based on ion intensities from full-scan MS/MS chromatograms. Chromatograms for all the compounds listed
in Table 1 were obtained in a single chromatographic run
at each concentration. Figure 2 shows reconstructed ion
chromatograms (RICs) from the analysis of the 50 pg/µL
standards. The MS/MS spectra for all the drugs, with the
exception of ketoprofen, are shown in Figure 3.
The MS/MS spectra were generated using a Normalized
Collision Energy of 28%. The use of Normalized Collision
Energy alleviates the necessity to optimize the collision
energy for each compound as is necessary in traditional
triple-quadrupole analysis, thus making this method
extremely easy to set up and run. Compounds that underwent a non-specific water loss were additionally fragmented using WideBand Activation (see Table 1). This
mode of fragmentation results in information-rich spectra
enabling structural confirmation without requiring an
additional MS3 transition. The compound ketoprofen
undergoes a neutral loss outside of the WideBand
Activation window and was selected for an MS/MS to
MS3 comparison study and is discussed later.
Chromatographic and mass spectrometric methods
were validated using the neat standards; subsequently the
experiments were repeated using standards in horse urine.
The RICs from these experiments are shown in Figure 4.
Using the RICs, calibration curves were created for each
of the compounds either neat (Figure 5) or in urine
matrix (Figure 6). The calibration curves were linear over
the three orders of magnitude assayed. In addition to
demonstrating linearity, the quantitative results shown in
Tables 2 and 3 demonstrate excellent reproducibility.
2
SEGMENT RT
1 3.40
4.44
4.56
2 5.58
5.71
6.02
6.20
6.49
3 7.20
4 8.95
9.60
10.16
5 11.28
11.33
11.93
12.05
ID#
COMPOUND
416
417
152
071
089
107
198
311
614
117
481
499
216
130
175
235
Theobromine
Theophylline
Dyphylline
Caffeine
Chlorothiazide
Cromolyn-Na
Hydroclorothiazide
Pemoline
Petoxifyline
Dexamethasone
Boldenone
Ketoprofen†
Indomethacin
Diclofenac
Flufenamic Acid
Meclofenamic Acid
M/Z
WB
181.0
181.0
255.1
195.1
-293.9
469.2
✔✔✔
-295.8
177.0
279.1
393.1
✔
287.1
✔
255 (209)
358.0
295.9
✔
282.1
295.9
✔
RIC
137 + 163 + 181
124 + 137
181
138
214 + 215
245
205 + 232 + 269
106
181
355 + 337 + 319
121 + 135 + 173
209 (105 + 194)
139 + 174
215 + 250
264
242 + 243
† Ketoprofen was analyzed by both MS/MS and MS3 for comparison study.
4 WB denotes use of wideband activation during MS/MS fragmentation.
Table 1: List of target compounds; corresponding RT (retention time), segment
(method segment see Figure 1), m/z denotes isolation mass and Ion Polarity,
WB–WideBand Activation, RIC–masses used in generation of Reconstructed
Ion Chromatograms for quantitation.
Figure 1: Instrument Setup settings for chlorothiazide (segment 2, scan event 2) and cromolyn-Na (segment 2, scan event 3)
RT: 2.99
100
RT: 5.97
100
50
50
50
RT: 4.25
0
Theobromine
Boldenone
0
Cromolyn-Na
0
RT: 4.24
100
RT: 9.60
100
RT: 10.15
100
RT: 6.08
100
50
50
Ketoprofen
50
RT: 3.01
Theophylline
0
100
Hydrochlorothiazide
0
100
RT: 4.36
50
0
100
RT: 6.45
50
Dyphylline
Pemoline
0
100
RT: 5.46
50
0
100
RT: 5.57
50
Flufenamic Acid
0
100
RT: 12.06
50
Meclofenamic Acid
Dexamethasone
Chlorothiazide
3
RT: 11.95
50
RT: 8.95
50
0
Diclofenac
0
100
Pentoxifylline
Caffeine
RT: 11.34
50
RT: 7.18
50
0
100
Indomethacin
0
100
50
0
100
RT: 11.28
0
4
5
Time (min)
0
6
6
7
8
Time (min)
9
10
11
Time (min)
12
Figure 2: Reconstructed ion chromatograms, using the product ions listed in Table 1 (RIC column), from the analysis of 10 µL of the 50 pg/µL
standards in solvent
Theobromine
Theophylline
162.97
100
80
137.01
60
40
Meclofenamic Acid
Dyphylline
123.95
100
180.93
100
Dexamethasone
242.95
100
355.03
100
80
80
80
60
60
60
40
40
40
80
241.92
60
337.03
319.08
40
139.88
180.99
20
157.88
110.01
100
20
197.79
0
150
m/z
200
Hydrochlorothiazide
100
163.02 181.03
150
m/z
200
100
150
200
20
100
250
177.30
150
181.03
200
m/z
250
200
300
80
80
60
60
60
60
60
40
40
40
100
150
20
231.94
159.78
0
295.88
200
m/z
250
100
300
Diclofenac
148.93
177.03
20
138.01
0
150
m/z
200
100
150
200
m/z
250
100
300
200
300
100
Cromolyn-Na
293.87
100
100
80
80
80
60
60
60
60
40
40
40
40
40
20
20
20
20
165.12
214.99
254.62
134.02
0
100
150
200
m/z
250
300
100
150
196.79
200
m/z
247.16
250
264.96
300
164.76
0
100
150
200
m/z
186.81
200
m/z
0
250
300
200
276.88
300
20
262.97
229.92
250
137.97
100
60
107.95
150
Caffeine
244.91
80
213.86
241.07
0
80
0
186.97
199.02
339.67
0
m/z
263.94
100
172.97
20
311.96
201.97
261.04
Chlorothiazide
Flufenamic Acid
249.86
100
221.01
135.02
40
138.88
20
135.86
0
400
Boldenone
100
80
20
300
m/z
80
204.92
356.96
0
174.00
100
237.03
148.92
249.92
227.82
Indomethacin
Pentoxifyline
100
309.10
20
150.22
0
m/z
105.93
100
236.99
219.04 240.10
130.08 156.03
0
80
40
Relative Abundance
137.02
Pemoline
268.88
100
20
119.98
0
300
m/z
424.89
505.47
400
500
109.93
138.98
177.00
195.01
0
100
150
m/z
200
Figure 3: Full-scan MS/MS spectra corresponding to compounds depicted in Figure 2
3
RT: 3.06
100
100
100
RT: 5.98
50
50
0
100
0
100
RT: 9.60
Cromolyn-Na
Theobromine
Boldenone
50
0
3.06
RT: 4.28
50
100
RT: 10.14
RT: 6.11
50
Hydrochlorothiazide
Theophylline
50
Ketoprofen
0
100
0
RT: 11.30
0
100
100
RT: 4.41
0
100
50
RT: 6.45
Pemoline (not found)
Dyphylline
50
0
100
50
RT: 11.35
Diclofenac
50
0
100
RT: 5.50
Indomethacin
RT: 7.17
0
Caffeine
50
Pentoxifylline
50
RT: 11.95
100
Flufenamic Acid
50
0
100
0
100
RT: 5.63
0
100
50
50
Chlorothiazide
Dexamethasone (not found)
0
0
3
4
5
RT: 12.07
50
Meclofenamic Acid
0
6
6
7
Time (min)
8
10
9
11
Time (min)
12
Time (min)
Figure 4: Reconstructed ion chromatograms, using the product ions listed in Table 1 (RIC column), from the analysis of a 10 µL injection of the 50 pg/µL
standard in horse urine
Y = 18.5962+131.036*X R^2 = 0.9986
25000
Theobromine
12000
Y = -233.655+246.415*X R^2 = 0.9994
Y = 4568.38+224.452*X R^2 = 0.9974
Y = -79.8728+249.537*X R^2 = 0.9998
14000
Theophylline
Theobromine
800000
20000
Theophylline
150000
6000
Area
600000
15000
Area
8000
Area
Area
10000
10000
400000
5000
200000
50000
4000
0
0
0
20
40
60
80
Y = -399.929+957.285*X R^2 = 0.9997
100000
20
40
60
80
100
80000
2000
3000
300000
20000
200000
20000
10000
60
80
100
Caffeine
500000
100000
0
0
0
20
40
60
Figure 5: Representative calibration curves from standards prepared
in solvent
80
300000
200000
0
40
600
Y = 1103.78+772.157*X R^2 = 0.9996
Dyphilline
100000
0
20
400
400000
40000
0
200
400000
30000
0
0
4000
500000
40000
Area
1000
Y = 9455.91+862.639*X R^2 = 0.9993
600000
Caffeine
50000
60000
0
Y = -604.499+521.811*X R^2 = 0.9991
Dyphilline
0
0
0
100
Area
2000
4
100000
200
400
600
0
200
400
100
Figure 6: Representative calibration curves from standards prepared
in horse urine
600
MS3 experiment was performed to generate additional
diagnostic ions without sacrificing sensitivity or reproducibility. To demonstrate this, standards and two urine QC samples
were analyzed in both MS/MS and MS3 mode, with results
shown in Figure 7. There is no loss of sensitivity, accuracy,
or reproducibility in obtaining this additional information.
The %RSD from the MS/MS and MS3 data are virtually
identical. While the sensitivity remains unchanged, the
accuracy in the analysis of the unknowns is actually
improved in the MS3 experiments (see Figure 7).
The %RSD for three replicate injections is less than 10% for
all neat standards at the 1 pg/µL level and higher (see Table 2).
The results for standards in urine were also excellent.
The %RSD, five replicate injections, for the lowest level
assayed in urine was less than 10% for most analytes (see
Table 3), and commonly less than 3% for mid- and
high-concentration samples. To complete the quantitative
study, two QC urine samples were analyzed. The results
shown in Table 4 demonstrate a high level of quantitation
accuracy, with a deviation of less than 10% for most analytes.
In addition, excellent reproducibility was demonstrated
with the %RSD being less than 8% for all but two
compounds (see Table 4).
Robustness
To assess the ruggedness of the method, a 166 pg/µL
standard in horse urine was assayed over 100 consecutive
injections. The results are displayed in Figure 9. The mean
and coefficient of variation (%CV) for four compounds:
theobromine, caffeine, pentoxyphylline, and ketoprofen
were determined to be less than 4% for all four compounds.
Ketoprofen – MS/MS vs. MS3: Ketoprofen undergoes a neutral
loss of a 46 amu fragment in MS/MS mode due to the loss of
the carboxyl group (see Ketoprofen structure). This is outside
of the mass window for WideBand Activation and thus, an
RT: 10.15
Relative Abundance
100
208.97
SN: 2857
100
100
SN: 2166
80
80
80
80
60
60
60
60
40
40
20
20
40
20
130.99
181.03
166.03
194.65
76.96
0
Time (min) 10.0
0
10.5
11.0
0
0
100
150
200
Time (min) 10.0
250
193.96
40
MS 3 : 255 209
MS/MS: 255
20
104.94
100
10.5
11.0
100
m/z
150
200
250
m/z
AREA
KETOPROFEN
CAL. CONC. %RSD
KETOPROFEN - MS3
AREA
CAL. CONC. %RSD
20350
19635
3.3 pg/µL 20281
20046
19185
6.073
5.751
6.042
5.936
5.548
8205
8012
3.75% 8201
7928
8137
3.874
3.636
3.87
3.531
3.79
45386
43015
16.6 pg/µL 44934
42855
42111
17.362
16.293
17.158
16.221
15.885
17551
18007
3.86% 17120
17380
17830
15.425
15.988
14.892
15.214
15.769
90353
87917
41.6 pg/µL 87285
91348
87594
37.637
36.539
36.254
38.086
36.393
37454
36469
2.23% 37773
36210
37289
40.022
38.805
40.417
38.485
39.818
2.09%
762536
760825
33.3 pg/µL 755161
762369
733111
340.72
339.948
337.394
340.644
327.452
279903
278910
1.67% 270466
282912
284341
339.663
338.436
328.
343.382
345.147
1.97%
1447518
1482572
650 pg/µL 1505908
1505854
1466808
649.573
665.378
675.901
675.876
658.27
554232
519271
1.72% 551821
552697
533792
678.703
635.495
675.723
676.806
653.441
Ketoprofen
Ketoprofen-MS 3
Y = 8058.18+2201.79*X R^2 = 0.9996
Y = 4911+812.486*X R^2 = 0.9993
600000
1500000
500000
Area
4.05%
400000
1000000
Area
SAMPLE
300000
200000
500000
100000
0
0
2.82%
0
200
400
0
600
200
41.6
41.6
38.0
41.7
91.4%
100.2%
1.22%
1.26%
600
Unknown 2
L)
µ
.
/
e
(pg
onc
enc
SD
fer
l. C
nc.
%R
Dif
Ca
Co
Unknown 1
L)
µ
.
/
e
(pg
onc
enc
SD
fer
l. C
nc.
%R
Dif
Ca
Co
Ketoprofen
Ketoprofen - MS3
400
104.1
104.1
104.6
106.1
100.5%
101.9%
CH3 O
CH – C – OH
O
C
2.86%
Figure 7: Comparison of MS/MS to MS3 quantitation of Ketoprofen
Ketoprofen
5
1.53%
2.05%
AVERAGE
AREA
AVERAGE
CALC.
CONC
%RSD
AVERAGE
AREA
0.1 pg/µL
Theobromine
Theophylline
Dyphylline
Caffeine
Chlorothiazine
Cromolyn-Na
Hydroclorothiazide
Pemoline
Petoxifyline
Dexamethasone
Boldenone
Ketoprofen
Indomethacin
Diclofenac
Flufenamic Acid
Meclofenamic Acid
AVERAGE
CALC.
CONC
%RSD
AVERAGE
AREA
0.5 pg/µL
72
0.49
0.52%
427
1
2.40%
176
0.25
42.33%
75
456
0
1
24.53%
1.28%
102
793
0.34
-0.32
18.81%
3.79%
219
315
-0.01
0.60
14.44%
7.64%
64
310
14
0.30
0.22
0.18
11.55%
13.97%
17.51%
400
4308
479
1261
1191
103
281
892
61
1
0
0
0
1
1
1
1
1
11.86%
1.86%
2.45%
1.97%
9.43%
11.11%
8.84%
2.52%
41.26%
234
784
149
917
255
780
8319
1017
2471
2381
212
475
1780
113
Table 2: Quantitation results for standards prepared in solvent
Theophylline (A)
Dyphylline (A)
Caffeine (A)
Chlorothiazine (A)
Hydroclorothiazide (A)
Pentoxifylline (A)
Boldenone (A)
Ketoprofen (A)
Ketoprofen – MS3 (A)
Indomethacin (A)
Diclofenac (A)
Meclofenamic Acid (A)
AVERAGE
AREA
AVERAGE
CALC.
CONC
3.3 pg/µL
1129
9832
6330
1798
2487
122524
13426
19899
8097
1087
2577
290
6
5
6
7
7
-2
7
6
4
2
3
7
%RSD
10.48%
1.94%
6.86%
5.99%
3.07%
-6.01%
2.77%
3.75%
4.05%
28.26%
2.37%
10.78%
AVERAGE
AREA
AVERAGE
CALC.
CONC
16.6 pg/µL
2540
22461
15401
3834
5684
296023
33942
43660
17578
2382
5355
447
17
17
18
17
18
16
19
17
15
13
13
10
6.6 pg/µL
Cromolyn-Na (B)
Flufenamic Acid (B)
11298
3442
9
15
6293
51
2.70%
3.59%
30183
7763
5185
44917
30016
7967
11276
605430
58628
88899
37039
6442
14471
2130
38
39
37
37
38
49
35
37
40
48
46
44
27
21
1.86%
17288
92
%RSD
AVERAGE
AREA
AVERAGE
CALC.
CONC
333.3 pg/µL
2.82%
0.98%
2.14%
0.94%
2.02%
2.36%
2.21%
2.23%
2.09%
4.05%
4.62%
2.92%
45170
322434
258749
70426
92748
3152583
593649
754801
279306
35792
78597
6086
334
334
336
334
331
332
334
337
339
332
332
122
83.3 pg/µL
1.08%
1.84%
88171
55407
100 pg/µL
Table 3: Quantitation results for standards prepared in horse urine
6
5.53%
2.73%
4.94%
5.48%
2.05%
3.35%
1.55%
3.86%
2.82%
6.78%
5.46%
22.75%
AVERAGE
CALC.
CONC
41.6 pg/µL
33.4 pg/µL
20 pg/µL
Theobromine (C)
%RSD
AVERAGE
AREA
83
88
51615
222
2.11%
2.24%
1.20%
1.24%
1.39%
0.97%
1.24%
1.67%
1.97%
2.61%
2.94%
16.84%
333.3 pg/µL
0.80%
2.44%
698392
135790
250 pg/µL
3.23%
%RSD
675
200
2.40%
13.92%
2000 pg/µL
1.58%
471898
2009
0.77%
AVERAGE
CALC.
CONC
%RSD
AVERAGE
AREA
1.0 pg/µL
AVERAGE
CALC.
CONC
%RSD
AVERAGE
AREA
5.0 pg/µL
AVERAGE
CALC.
CONC
%RSD
AVERAGE
AREA
10 pg/µL
AVERAGE
CALC.
CONC
%RSD
AVERAGE
AREA
50 pg/µL
AVERAGE
CALC.
CONC
%RSD
100 pg/µL
1
1
1.79%
3.10%
625
1208
4359
2330
5
5
5
6
0.47%
2.58%
0.91%
5.03%
1222
2408
8593
3912
9
10
9
9
2.55%
2.52%
0.72%
3.15%
6856
12207
46667
26131
52
49
49
51
0.41%
1.36%
1.47%
0.85%
12993
24967
95782
51308
99
100
100
99
1.43%
0.66%
1.13%
1.07%
1
1
1
1
1
1
1
1
1
1
1
1
22.14%
5.66%
7.88%
3.65%
1.13%
5.52%
5.08%
7.15%
2.78%
8.40%
6.92%
7.84%
645
4672
1170
3774
44808
5758
12325
11906
212
2382
8546
641
4
5
5
5
5
5
5
5
1
5
5
5
13.31%
7.11%
11.66%
2.79%
0.56%
1.14%
2.25%
1.22%
2.78%
1.20%
0.39%
7.97%
1294
8889
2663
8117
93532
11952
23958
22945
2259
4712
17468
1446
9
9
10
10
10
10
10
10
10
10
10
10
4.88%
2.63%
2.83%
1.35%
1.05%
0.47%
1.07%
3.13%
2.80%
3.18%
2.60%
12.83%
7711
49369
13545
42321
474442
60483
120812
119082
11565
24920
90104
7294
53
51
50
50
53
51
50
48
50
50
50
51
2.66%
3.40%
1.19%
0.62%
2.08%
2.69%
1.33%
1.17%
1.77%
1.14%
0.36%
1.39%
14231
96148
26856
84890
877603
118294
238560
253903
23189
50161
178996
14337
98
100
100
100
98
100
100
101
100
100
100
100
1.10%
1.12%
2.72%
1.79%
1.77%
1.68%
1.68%
0.46%
0.18%
2.06%
1.28%
0.21%
AVERAGE
AREA
96129
575760
511382
143677
186723
5840616
1301762
1481732
542362
61378
122821
7065
AVERAGE
CALC.
CONC
650 pg/µL
667
667
666
666
668
667
666
665
664
667
668
142
%RSD
1.31%
3.71%
1.21%
0.54%
1.64%
0.95%
1.22%
1.72%
2.86%
1.74%
2.18%
10.74%
QC Sample
)
.
/µL
e
g
p
(
onc
enc
SD
fer
l. C
nc.
%R
Dif
Ca
Co
1350 pg/µL
88842
170496
84
249
2.10%
10.77%
4000 pg/µL
825323
3998
1.85%
Theobromine (C) 250.0
Theophylline (A) 41.6
Dyphylline (A) 41.6
Caffeine (A) 41.6
Chlorothiazine (A) 41.6
Cromolyn-Na (B) 83.3
Hydroclorothiazide (A) 41.6
Pentoxifylline (A) 41.6
Boldenone (A) 41.6
Ketoprofen (A) 41.6
Ketoprofen – MS3 (A) 41.6
Indomethacin (A)
Diclofenac (A)
Flufenamic Acid (B)
Meclofenamic Acid (A)
41.6
41.6
83.3
41.6
QC Sample 2
)
L
µ
.
/
e
(pg
onc
enc
SD
fer
l. C
nc.
%R
Dif
Ca
Co
231.7
38.6
41.3
42.4
43.0
83.9
41.8
44.5
38.8
38.0
41.7
92.7%
92.7%
99.3%
101.9%
103.3%
100.7%
100.5%
106.9%
93.4%
91.4%
100.2%
1.72%
1.96%
2.02%
3.30%
2.64%
2.10%
2.64%
2.23%
1.04%
1.22%
1.26%
625.0
104.1
104.1
104.1
104.1
210.0
104.1
104.1
104.1
104.1
104.1
615.0
103.5
115.5
109.6
114.4
193.9
113.1
126.5
102.4
104.6
106.1
98.4%
99.4%
110.9%
105.3%
109.9%
92.4%
108.6%
121.5%
98.4%
100.5%
101.9%
1.84%
2.58%
3.38%
3.05%
1.65%
1.77%
2.27%
1.43%
2.63%
1.53%
2.05%
49.7
48.4
60.7
33.3
119.5%
116.3%
72.9%
80.1%
5.78%
5.89%
2.21%
6.74%
104.1
104.1
210.0
104.1
116.4
124.1
141.6
89.8
111.8% 1.62%
119.2% 7.94%
67.4% 19.10%
86.3% 21.76%
Table 4: Quantitation results for the analysis of unknown levels of drugs in horse urine
7
achieve the low % RSD required in quantitation due to
the fast cycle time of the Thermo Scientific LTQ. In the
case of non-specific neutral molecule losses, MS3
experiments generated diagnostic spectra for
confirmational purposes while providing quantitative
results comparable to the MS/MS data. Results of the
ruggedness study demonstrate no appreciable loss of
sensitivity or reproducibility across 100 replicate urine
injections. Thus, using the Thermo Scientific LTQ
two-dimensional linear ion trap, we have demonstrated
the development of a simple, rapid, and rugged method
capable of confirmational screening and simultaneous
quantitation of drugs in horse urine using both full-scan
LC/MS/MS and MS3 spectra.
Conclusions
Positive and negative ion detection of co-eluting drugs
was accomplished in a single chromatographic run
using automated polarity switching. Drugs that
underwent a neutral water loss were further fragmented
using WideBand Activation to provide a diagnostically
rich MS/MS spectrum for structural confirmation.
The compound ketoprofen underwent a prominent,
non-specific neutral loss of formic acid and was further
analyzed using an MS3 transition. Full-scan MSn data
was reprocessed to quantify all 16 compounds by
reconstructed ion chromatograms (RICs), or postacquisition MRM, and provided results comparable to
triple quadrupole SRM quantitation. It is possible to
45000
40000
B
35000
35000
30000
30000
25000
25000
20000
Average Area = 18574
CV = 3.43%
40000
Area
Area
Average Area = 27690
CV = 2.32%
A
20000
15000
15000
10000
10000
5000
5000
0
0
0
20
40
60
80
100
0
20
Injection Number
Average Area = 482146
CV = 3.81%
1000000
C
900000
800000
600000
500000
Area
Area
700000
400000
300000
200000
100000
0
0
20
40
40
60
80
100
Injection Number
60
Injection Number
80
100
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
Average Area = 65211
CV = 2.41%
D
0
20
40
60
80
100
Injection Number
Figure 8: Ruggedness and reproducibility for 100 consecutive injections of a 166 pg/µL standard of theobromine, caffeine, pentoxyphylline,
and ketoprofen in urine
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