Page 217 - CRFTApplication Compendium_090613

3 Thermo Scienti c Poster Note • PN63783_E 03/13S
dilution method. When a lower limit of
traction was developed to concentrate
inearity and detection limits, but also
rd responses. To investigate and
evaporation step, the following
samples for either 15 minutes or
bes before evaporation to prevent
15 or 60 minutes; 5) Add 20 µL DMSO
lity is an issue; and 6) Spike a blank
on as 100% recovery. Results shown in
l, especially for mephedrone, the
e an issue for naphyrone.
the forensic toxicological analysis of the
and mephedrone, as well as other
butylone and naphyrone in urine with
PLC/MS/MS analysis on a Thermo
ss spectrometer.
good reproducibility and accuracy for
hylone and butylone. Naphyrone
litative using this method.
come the latest abused designer
in the African Catha edulis (khat) plant,
hetamine- and cocaine-like effects. As
n base structure abound (Figure 1). On
cement Agency (US DEA) listed three of
pyrovalerone (MDPV), methylone and
king them illegal. As these drugs are
ests, new methods are needed to detect
FIGURE 3. HPLC gradient for
cathinone analysis.
FIGURE 4. Mass spectrometer
source conditions.
Data Analysis
Data acquisition and processing wer
TraceFinder™ software.
Validation
Standard curves were prepared by f
Quality control (QC) samples were p
(MQC) and high (HQC) concentratio
determined by processing six replica
as outlined in the Sample Preparatio
were investigated by comparing pea
standard prepared in 12 different lot
FIGURE 2. Results for method development experiments to determine effects of
evaporation step in sample processing. P = plain tubes after -15 and -60 min
evaporation; DMSO = tubes with DMSO added prior to -15 and -60 min
evaporation; PD = evaporated without DMSO, add DMSO after 15 min
evaporation; Spike = compounds spiked after 15 min evaporation
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
Mephedrone 178.1226
Mephedrone-d3 181.1415
Methylone 208.0968
Methylone-d3 211.1156
MDPV 276.1594
MDPV-d8 284.2096
Naphyrone 282.1852
Ethylone 222.1125
Ethylone-d5 227.1438
Butylone 222.1125
Butylone-d3 225.1313
Methedrone 194.1176
Time
(min) %A %B %C Flow(µL/min)
0 90 10 0 500
0.15 90 10 0 500
2.15 5 95 0 500
2.45 5 95 0 500
2.46 0 0 100 500
3.30 0 0 100 500
3.31 90 10 0 500
5.00 90 10 0 500
FIGURE 6. Exact masses and nor
FIGURE 5. Diagram of Q Exactive
Sample Preparation
Deuterated internal standards were available for all compounds except methedrone
and naphyrone. Butylone-d3 was used as internal standard for methedrone and
MDPV-d8 was used for naphyrone. Samples preparation is a liquid-liquid extraction
(LLE). 200 µL of urine and 10 µL of internal standard mix solution (2 µg/mL of each
deuterated IS) were basified with 100 µL of 1 N NaOH. Extraction was performed by
adding 1 mL of ethylacetate:hexane (1:1), mixing and centrifuging. 800 µL of the
resulting supernatant was transferred to a clean test tube containing 20 µL of DMSO to
prevent complete evaporation of solvent. Analytes are small and slightly volatile, and
will evaporate if left too long in the evaporator. Solvent was evaporated at 37 °C under
nitrogen for 15 minutes. 200 µL of 5% methanol was added, mixed and transferred to
an HPLC vial with limited-volume insert. 20 µL was then injected onto HPLC-MS.
Liquid Chromatography
Chromatographic analysis was performed using the Thermo Scientific™Accela™ 600
HPLC pump and a Thermo Scientific™ Hypersil™ GOLD C18 column (50 x 2.1 mm,
3 µm particle size) under gradient conditions (Figure 3). Mobile phases A and B
consisted of 10 mM ammonium formate with 0.1% formic acid in water and methanol,
respectively. Mobile phase C was acetonitrile:1-propanol:acetone (45:45:10). The total
run time was 5 minutes.
Mass Spectrometry
MS analysis was carried out on a Thermo Scientific Q Exactive bench-top Orbitrap
mass spectrometer equipped with a heated electrospray ionization (HESI-II) probe
(Figure 5). The Q Exactive was operated in t-MS2 mode at a resolution of 17,500
(@m/z=200). Exact masses, collision energies and fragment ions are listed in Figure 6.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Peak Area Relative to Spiked Sample
butylone
butylone-d3
ethylone
ethylone-d5
MDPV
MDPV-d8
mephedrone
meph-d3
methedrone
methylone
methy-d3
naphyrone
Results
MDPV, methylone, mephedrone, m
from 0.5 to 1000 ng/mL. Figure 7 sh
compounds. Figure 8 shows repres
compounds tested. Inter-assay qual
the method to be reproducible acro
Limited matrix effects were seen for
mediated by deuterated internal sta
tested in various lots of urine comp
89% to 163%. Relative recoveries r
lots also improved when deuterated
s
NH CH 3
CH3
CH
3
O
mephedrone
naphyrone
butylone e
H3
NH CH3 O
O
NH CH
3
O
CH3
O
N
CH3