1. Liquid Chromatograph Mass Spectrometer
SSI-LCMS-060
Liquid Chromatography Mass Spectrometry
Analysis of 90 Multi-Class Drugs
with Polarity Switching in Plasma
Summary: Ninety drugs and internal
standards were prepared in plasma using a
Biotage SLE 96-well plate and analyzed on an
LCMS-8050 .
Background: Plasma is an important matrix to
measure the exposure of the drug prior to
elimination. Clinical research often requires
that drug concentrations in blood be
monitored. The capability of the LCMS-8050 to
detect low concentrations of drugs in plasma
was evaluated.
Method:
A 200 µL aliquot of plasma was extracted
using a Biotage SLE 96-well plate. Samples
were diluted with H2O and eluted using ethyl
acetate. The eluent was then dried down
under nitrogen gas on an SPE dry 96
evaporation system and reconstituted in
mobile phase, all in a 96-well plate. Samples
were analyzed on an LCMS-8050 in MRM
mode with a Nexera MP front end. Drugs were
separated using a Biphenyl column (2.7uM
,100 X 2.1mm) from Restek. All analytes were
eluted in under 4 minutes.
Figure 1. Chromatogram of all analytes eluted in under 4 minutes.

2. SSI-LCMS-060
0.75 1.00
0
1000
2000
3000
4000
5000
6000
7000 136.10>119.05(+)
136.10>90.85(+)
1.00 1.25
0
25000
50000
75000
328.10>212.20(+)
328.10>310.10(+)
Amphetamine
Naloxone
2.00 2.25
0
1000
2000
3000
4000
5000
6000
264.15>42.10(+)
264.15>58.00(+)
Tramadol
2.50 2.75
0
250
500
750
1000
1250
1500
1750 468.30>396.20(+)
468.30>55.10(+)
Buprenorphine
2.00 2.25
0
250
500
750
230.90>85.10(-)
231.00>42.10(-)
Diazapam
Phenobarbital
THC-COOH
3.75 4.00
0
10000
20000
30000
40000
285.10>257.10(+)
285.10>221.90(+)
4.00 4.25
0
500
1000
1500
2000
2500
3000
3500 345.05>193.10(+)
345.05>299.00(+)
3.75 4.00
0
5000
10000
15000
20000
344.20>155.10(+)
344.20>127.10(+)
JWH-073-(3-OH-butyl)
1.00 1.25
0
1000
2000
3000
4000
302.10>227.00(+)
302.10>198.10(+)
Fentanyl
Zopiclone
Oxymorphone
2.00 2.25
0
1000
2000
3000
4000
5000
6000
7000
389.10>216.80(+)
389.10>244.80(+)
2.25 2.50
0
1000
2000
3000
4000
5000 337.25>188.10(+)
337.25>105.00(+)
3.00 3.25
0
10000
20000
30000
40000
50000
60000 299.90>282.50(+)
299.90>227.00(+)
Chlordiazepoxide
Figure 2. Chromatograms at the LLOQ for representative analytes.

3. 0 100 200 300 Conc. Ratio
0.0
0.5
1.0
1.5
2.0
Area Ratio
0.00 0.05 0.10 0.15 Conc. Ratio
0.000
0.025
0.050
0.075
0.100
0.125
0.150
Area Ratio
0.0 0.5 1.0 1.5 Conc. Ratio
0.00
0.25
0.50
0.75
Area Ratio
0 250 500 750 Conc. Ratio
0.00
0.05
0.10
0.15
Area Ratio
0.0 25.0 50.0 75.0 Conc. Ratio
0.00
0.25
0.50
0.75
Area Ratio
0.0 0.5 1.0 1.5 Conc. Ratio
0.00
0.25
0.50
Area Ratio
0.0 0.5 1.0 1.5 Conc. Ratio
0.00
0.05
0.10
0.15
Area Ratio
0.0 0.1 0.2 0.3 Conc. Ratio
0.00
0.05
0.10
0.15
0.20
Area Ratio
0 250 500 750 Conc. Ratio
0
5
10
15
20
25
Area Ratio
0.0 0.5 1.0 1.5 Conc. Ratio
0.000
0.025
0.050
0.075
Area Ratio
Amphetamine
R2 = 0.993
Naloxone
SSI-LCMS-060
Oxymorphone
R2 = 0.998
THC-COOH
Phenobarbital
R2 = 0.995
R2 = 0.998
R2 = 0.996
R2 = 0.997
R2 = 0.994
R2 = 0.996R2 = 0.996
ZopicloneDiazepam
Tramadol
Buprenorphine
Fentanyl
JWH-073-(3-OH-butyl) Chlordiazepoxide
0 100 200 300 Conc. Ratio
0.0
0.5
1.0
1.5
2.0
Area Ratio
0.0 25.0 50.0 75.0 Conc. Ratio
0.0
0.1
0.2
0.3
0.4
0.5
Area Ratio
R2 = 0.995
R2 = 0.995
R2 = 0.996
Figure 3. Calibration curves for representative analytes.

4. SSI-LCMS-060
Name
Retention
time
Area
Linear Range
(ng/mL)
Calc. Con.
(ng/mL)
Std. Conc. Accuracy
Morphine 0.619 135853 25 - 500 24.42 25 97.7
Oxymorphone 1.183 26554 5 - 500 5.02 5 100.4
Noroxycodone 0.649 43649 2.5 - 250 2.63 2.5 105.1
Hydromorphone 0.711 156603 5 - 250 5.08 5 101.5
Amphetamine 0.985 33637 5 - 500 4.88 5 97.5
Methylhexanamine 1.065 101215 5 - 500 5.2 5 103.9
Naloxone 1.108 504071 10 - 1000 10.09 10 100.9
6-MAM 1.278 5731 0.4 - 40 0.39 0.4 98.7
Methamphetamine 1.209 16449 5 - 500 5.13 5 102.6
Phentermine 1.214 7,750 5 - 500 5.18 5 103.6
Codeine 1.249 50918 5 - 500 5.1 5 102
Norhydrocodone 1.259 107,903 5 - 500 5.17 5 103.4
Methylone 1.277 236363 1 - 100 1.02 1 102.3
O-desmethyltramadol 1.307 364833 5 - 500 5.37 5 107.4
MDA 1.21 172644 5 - 500 5.19 5 103.8
4-methylephedrine 1.359 80,290 5 - 500 5.23 5 104.6
Oxycodone 1.414 176617 2.5 - 250 2.54 2.5 101.7
MDMA 1.442 419135 5 - 500 5.06 5 101.2
Hydrocodone 1.493 20536 5 - 500 5.02 5 100.4
Diethylpropion 1.636 13,651 5 - 500 5.09 5 101.8
MDEA 1.711 236144 5 - 500 5.05 5 101
Norfentanyl 1.926 23074 0.4 - 40 0.41 0.4 102.3
Tapentadol 1.954 30340 2.5 - 250 2.58 2.5 103.3
Dextrorphan 1.999 299180 5 - 500 5.28 5 105.5
Tramadol 2.044 29628 1 - 100 1.05 1 104.9
Benzoylecgonine 2.083 407 2.5 - 250 2.53 2.5 101.1
Methylphenidate 2.085 100482 5 - 500 4.64 5 92.9
Normeperidine 2.105 166951 2.5 - 250 2.64 2.5 105.7
alpha-PVP 2.113 85311 1 - 100 1.04 1 104.3
Meperidine 2.119 192731 2.5 - 250 2.55 2.5 102.1
Meprobamate 2.142 90452 5 - 500 5.31 5 106.2
Bupropion 2.151 81502 2.5 - 100 2.56 2.5 102.4
Zopiclone 2.165 36901 1 - 100 1.04 1 103.6
MDPV 2.8 145770 1 - 100 1.02 1 102
Phenobarbital 2.243 4155 10 - 1000 10 10 100
7-aminoclonazepam 2.244 347504 4 - 400 4.04 4 101
Norbuprenorphine 2.256 2335 2.5 - 250 2.45 2.5 97.9
Butalbital 2.305 4730 10 - 1000 10.37 10 103.7
Desmethyldoxepin 2.448 272384 5 - 500 5.31 5 106.2
Zolpidem 2.454 24029 1 - 20 1.04 1 103.8
Pentobarbital 2.461 7623 10 - 200 10.07 10 100.7
Amobarbital 2.464 5103 10 - 200 10.33 10 103.3
Doxepin 2.477 19875 5 - 100 5.14 5 102.9
Fentanyl 2.494 26377 0.05 - 5 0.05 0.05 103.2
Fluoxetine 2.503 474850 2.5 - 50 2.66 2.5 106.6
Buprenorphine 2.553 7540 0.5 - 10 0.5 0.5 100.9
Secobarbital 2.578 4,379 10 - 200 10.23 10 102.3
Flurazepam 2.584 90,697 4 - 80 4.11 4 102.80
Mitragynine 2.78 2624 0.1 - 2 0.1 0.1 102.4
Carisoprodol 2.601 191006 5 - 100 5.13 5 102.6
Phencyclidine 2.615 35040 0.5 - 10 0.5 0.5 100.6
Propoxyphene 2.663 76949 5 - 100 5.04 5 100.7
Protriptyline 2.712 12487 5 - 500 4.9 5 98
Desipramine 2.722 190852 5 - 500 5.14 5 102.8
Table 1. The linear range and results for the LLOQ of all analytes.

5. SSI-LCMS-060
Name Retention time Area Linear Range(ng/mL) Calc. Con. (ng/mL)
Std.
Conc.
Accuracy
Imipramine 2.774 124106 5 - 100 5.09 5 101.8
Cyclobenzaprine 2.801 292087 5 - 250 5.06 5 101.1
EDDP 2.81 64096 5 - 100 5.21 5 104.2
Nortriptyline 2.82 22163 5 - 500 4.85 5 96.9
Amitriptyline 2.853 517 5 - 500 4.83 5 96.6
Trimipramine 2.934 105597 5 - 500 5.07 5 101.5
N-
desmethylflunitrazepam
2.942 156263 4 - 200 3.94 4 98.4
Lorazepam 2.995 68387 4 - 200 4.13 4 103.3
Desmethylclomipramine 3.035 93075 5 - 250 5.25 5 105
Risperidone 2.539 164080 0.5 - 10 0.53 0.5 105.3
Chlordiazepoxide 3.055 335299 5 - 500 4.93 5 98.5
Oxazepam 3.07 61690 4 - 400 4.03 4 100.7
Clonazepam 3.082 123232 4 - 400 3.96 4 99
Methadone 3.086 18359 12.5 - 250 12.76 12.5 102.1
Clomipramine 3.082 162619 5 - 500 5.08 5 101.7
Triazolamx 3.181 47530 4 - 200 4.09 4 102.2
Desalkylflurazepam 3.183 76196 4 - 400 4.13 4 103.1
Midazolamx 3.223 276869 4 - 400 4.2 4 105
Midazolam 3.255 539866 4 - 200 4.17 4 104.2
Alprazolax 3.257 108930 4 - 400 4.16 4 103.9
Methaqualone 3.296 73990 10 - 500 10.44 10 104.4
Zaleplon 3.327 587109 5 - 100 4.57 5 91.5
Nordiazepam 3.406 162996 4 - 400 4.17 4 104.3
Flunitrazepam 3.467 298163 4 - 400 4.22 4 105.5
Triazolam 3.539 263725 4 - 80 4.08 4 102.1
Temazepam 3.825 1422 10 - 400 10.49 10 104.9
Alprazolam 3.628 81689 4 - 400 3.98 4 99.6
AB-PINACA 3.776 413805 1.5 - 150 1.54 1.5 102.6
Diazepam 3.91 176095 4 - 400 4 4 99.9
JWH-073-(3-OH-butyl) 3.939 78981 1 - 100 1.03 1 102.6
AB-FUBINACA 3.64 243101 1.5 - 150 1.55 1.5 103.5
JWH-018-(4-OH-pentyl) 4.093 19287 1 - 100 1.02 1 102.4
THC-COOH 4.212 11899 1.5 - 150 1.47 1.5 97.8
XLR-11 4.35 53481 3.75 - 75 3.86 3.75 103
Aripiprazole 3.23 3625 1 - 50 1 1 99.7
Quetapine 2.698 21954 2.5 - 250 2.49 2.5 99.5
Table 1.(Continued) The linear range and results for the LLOQ of all analytes.

6. SSI-LCMS-060
Results and Discussion: Ninety different
drugs and internal standards from multiple
drug classes including opioids, amphetamines,
sympathomimetics, benzodiazepines,
barbiturates, tricyclic antidepressants, and
cannabinoids were extracted from plasma.
With an extraction time under an hour, the SLE
extraction in the 96 well format provides a high
throughput sample prep technique to
compliment the ultra fast separations and scan
speeds of the LCMS-8050 coupled to a Nexera
LC system.
Figure 1 shows representative
chromatograms at the LLOQ for a few analytes
demonstrating excellent signal to noise. These
analytes represent the many drug classes that
can be detected and quantitated by the LCMS-
8050. As shown in Table 1 and Figure 2., the
LCMS-8050 demonstrated excellent accuracy
and linearity. Accuracy at the LLOQ ranged
from 96.6% to 107.4%. The R2 value for all
curves was >0.990.
Conclusion: Using a rapid chromatographic
separation, 90 drugs were detected and
quantitated in less than five minutes. The
LCMS-8050 was demonstrated to provide
highly sensitive and accurate detection of
drugs in plasma extracts.