Page 218 - CRFTApplication Compendium_090613

4 Quantitation of Seven Designer Cathinones in Urine Using Q Exactive Mass Spectrometer
FIGURE 4. Mass spectrometer
source conditions.
Data Analysis
Data acquisition and processing were performed using Thermo Scientific™
TraceFinder™ software.
Validation
Standard curves were prepared by fortifying pooled blank human urine with analytes.
Quality control (QC) samples were prepared in a similar manner at low (LQC), middle
(MQC) and high (HQC) concentrations. Intra-run variability and robustness were
determined by processing six replicates of each QC level along with a calibration curve
as outlined in the Sample Preparation section on three different days. Matrix effects
were investigated by comparing peak areas of analyte at 10 ng/mL, and internal
standard prepared in 12 different lots of urine to those of a sample prepared in water.
pment experiments to determine effects of
g. P = plain tubes after -15 and -60 min
O added prior to -15 and -60 min
t DMSO, add DMSO after 15 min
ked after 15 min evaporation
Although naphyrone was det
other compounds and a gre
naphyrone ranged from 146
internal standard ranged fro
tried, and MDPV-d8 showed
naphyrone does not allow fo
precision. In this assay, nap
Parameter Value
Sheath Gas 35
Aux gas 15
Sweep gas 1
Discharge current 4
Capillary temp 320
S-Lens RF Level 60
Vaporizer Temp 350
Analyte m/z NCE Quantifier m/z Qualifier m/z
Mephedrone 178.1226 35% 160.1119 145.0885
Mephedrone-d3 181.1415 35% 163.1306 148.1072
Methylone 208.0968 35% 160.0756 190.0861
Methylone-d3 211.1156 35% 163.0943 193.1049
MDPV 276.1594 60% 126.1278 135.0440
MDPV-d8 284.2096 60% 134.1779 135.0440
Naphyrone 282.1852 45% 141.0697 211.1115
Ethylone 222.1125 45% 174.0911 204.1016
Ethylone-d5 227.1438 45% 179.1224 209.1329
Butylone 222.1125 45% 174.0912 204.1017
Butylone-d3 225.1313 45% 177.1099 207.1205
Methedrone 194.1176 35% 176.1069 161.0834
)
FIGURE 6. Exact masses and normalized collision energies (NCE) for cathinones
FIGURE 5. Diagram of Q Exactive Mass Spectrometer
FIGURE 7. Representative
0
1
2
3
4
5
6
7
8
9
10
0 200 400 600
Area Ratio
ng/mL
butylone
R2 = 0.9969
0
1
2
3
4
5
6
7
8
9
10
0 200 400 600
Area Ratio
ng/mL
MDPV
R2 = 0.9930
0
2
4
6
8
10
12
0 200 400 600
Area Ratio
ng/mL
methedrone
R2 = 0.9950
0
1
2
3
4
5
6
0 200 400 600
Area Ratio
ng/mL
naphyrone
R 2 = 0.9805
ilable for all compounds except methedrone
s internal standard for methedrone and
ples preparation is a liquid-liquid extraction
nal standard mix solution (2 µg/mL of each
of 1 N NaOH. Extraction was performed by
), mixing and centrifuging. 800 µL of the
a clean test tube containing 20 µL of DMSO to
t. Analytes are small and slightly volatile, and
orator. Solvent was evaporated at 37 °C under
ethanol was added, mixed and transferred to
20 µL was then injected onto HPLC-MS.
d using the Thermo Scientific™Accela™ 600
Hypersil™ GOLD C18 column (50 x 2.1 mm,
tions (Figure 3). Mobile phases A and B
with 0.1% formic acid in water and methanol,
nitrile:1-propanol:acetone (45:45:10). The total
o Scientific Q Exactive bench-top Orbitrap
ted electrospray ionization (HESI-II) probe
in t-MS2 mode at a resolution of 17,500
ergies and fragment ions are listed in Figure 6.
butylone
butylone-d3
ethylone
ethylone-d5
MDPV
MDPV-d8
mephedrone
meph-d3
methedrone
methylone
methy-d3
naphyrone
Results
MDPV, methylone, mephedrone, methedrone, ethylone and butylone were all linear
from 0.5 to 1000 ng/mL. Figure 7 shows representative calibration curves for all
compounds. Figure 8 shows representative chromatogram at 0.5 ng/mL for all
compounds tested. Inter-assay quality control statistics shown in Figure 9 demonstrate
the method to be reproducible across the calibration range for the above compounds.
Limited matrix effects were seen for the above compounds, and those were largely
mediated by deuterated internal standards. The absolute recoveries of all cathinones
tested in various lots of urine compared to a sample prepared in water ranged from
89% to 163%. Relative recoveries ranged from 105% to 136%. Precision across all
lots also improved when deuterated internal standards were used.