Page 216 - CRFTApplication Compendium_090613

2 Quantitation of Seven Designer Cathinones in Urine Using Q Exactive Mass Spectrometer
Methods
Method Development
This assay was originally developed as a urine dilution method. When a lower limit of
quantitation (LOQ) was desired, liquid/liquid extraction was developed to concentrate
the samples. Initial experiments showed good linearity and detection limits, but also
low recovery and highly variable internal standard responses. To investigate and
mediate the possible loss of analytes during the evaporation step, the following
experiments were performed: 1 & 2) Evaporate samples for either 15 minutes or
60 minutes; 3 & 4) Add 20 µL of DMSO to the tubes before evaporation to prevent
samples from evaporating to dryness, again for 15 or 60 minutes; 5) Add 20 µL DMSO
to tubes after evaporation to determine if solubility is an issue; and 6) Spike a blank
processed sample with analytes after evaporation as 100% recovery. Results shown in
Figure 2 indicate that evaporation time is critical, especially for mephedrone, the
smallest molecule tested, and solubility might be an issue for naphyrone.
Overview
Purpose: To develop an HPLC-MS method for the forensic toxicological analysis of the
three Schedule I cathinones: MDPV, methylone and mephedrone, as well as other
substituted cathinones: methedrone, ethylone, butylone and naphyrone in urine with
limits of quantitation (LOQs) of 0.5 ng/mL.
Methods: Liquid/liquid extraction followed by HPLC/MS/MS analysis on a Thermo
Scientific™ Q Exactive™ benchtop Orbitrap mass spectrometer.
Results: We achieved LOQs of 0.5 ng/mL with good reproducibility and accuracy for
MDPV, mephedrone, methylone, methylone, ethylone and butylone. Naphyrone
showed more variability and is considered qualitative using this method.
Introduction
Substituted cathinones, or “Bath Salts,” have become the latest abused designer
drugs. Based on cathinone, a substance found in the African Catha edulis (khat) plant,
substituted cathinones are stimulants with amphetamine- and cocaine-like effects. As
with many designer drugs classes, variations on base structure abound (Figure 1). On
October 21, 2011 the United States Drug Enforcement Agency (US DEA) listed three of
the most common chemicals – methylenedioxy pyrovalerone (MDPV), methylone and
mephedrone – as Schedule I drugs, thereby making them illegal. As these drugs are
not detected by current ELISA drug screening tests, new methods are needed to detect
and quantitate these compounds.
FIGURE 3. HPLC gradient for
cathinone analysis.
FIGURE 2. Results for method
evaporation step in sample pr
evaporation; DMSO = tubes w
evaporation; PD = evaporated
evaporation; Spike = compou
Time
(min) %A %B %C (
0 90 10 0
0.15 90 10 0
2.15 5 95 0
2.45 5 95 0
2.46 0 0 100
3.30 0 0 100
3.31 90 10 0
5.00 90 10 0
Sample Preparation
Deuterated internal standards w
and naphyrone. Butylone-d3 wa
MDPV-d8 was used for naphyro
(LLE). 200 µL of urine and 10 µ
deuterated IS) were basified wit
adding 1 mL of ethylacetate:hex
resulting supernatant was transf
prevent complete evaporation of
will evaporate if left too long in t
nitrogen for 15 minutes. 200 µL
an HPLC vial with limited-volum
Liquid Chromatography
Chromatographic analysis was
HPLC pump and a Thermo Scie
3 µm particle size) under gradie
consisted of 10 mM ammonium
respectively. Mobile phase C wa
run time was 5 minutes.
Mass Spectrometry
MS analysis was carried out on
mass spectrometer equipped wi
(Figure 5). The Q Exactive was
(@ m/z=200). Exact masses, col
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Peak Area Relative to Spiked Sample
FIGURE 1. Structures of designer cathinones
NH CH 3
CH3
CH
3
O
NH CH3
CH 3
O
O
CH
3
O
O CH 3
NH CH
3
O
mephedrone
methylone
naphyrone
butylone ethylone
methedrone
MDPV
O
O CH3
NH
O
CH3 O
O
NH CH
3
O
CH3
O
N
CH3
O
O
N
O
CH3