Page 338 - CRFTApplication Compendium_090613

3. Mass Spectrometer Conditions
MS analysis was carried out on a MSQ Plus single
quadrupole LC-MS detector (Thermo Scientific, San Jose,
CA). The MS conditions were as follows:
Ionization: Electrospray (ESI)
Polarity: Positive
Probe Temperature: 450 °C
Cone Voltage: 60 V
Scan Mode: Full scans (100-500 m/z) and/or Selected ion
monitoring (SIM)
ESI Voltage: 4.5 kV
Results and Discussion
1. MS Detection
Both positive and negative electrospray analysis were
performed using the polarity switch function of the
Xcalibur software. All of the analytes exhibited higher
ionization efficiency in the positive ion mode compared
with the negative mode. The MS spectra of the drug
standards show both molecular ion signals of [M+H]+ and
acetonitrile adducts of the form [M+ACN+H]+. For 13 of
the analytes, the signal from the molecular ion was more
intense than the signal from the acetonitrile adduct.
For amphetamine, the most intense signal was from the
acetonitrile adduct [M+ACN+H]+ at m/z of 177.2 (data
not shown).
2. Separations with Standard Stationary Phases
Three columns were evaluated to separate the illicit drug
mixtures: Hypersil GOLD, Hypersil GOLD aQ and
Hypersil GOLD PFP (Figure 1). The UHPLC method with
each column type was optimized individually. Hypersil
GOLD aQ, a polar endcapped C18 phase which offers
more retention of polar compounds, did not resolve the
early eluting compounds including methamphetamine,
oxycodone, caffeine, MDMA and hydrocodone.3 The
separation on Hypersil GOLD aQ may have been
impaired by interactions between the polar endcapped
stationary phase and the polar analytes. Hypersil GOLD,
with LI or C18 selectivity, showed improved selectivity for
all analytes except caffeine (peak 1) and oxycodone (peak 7).
Hypersil GOLD uses highly pure silica and endcapping
procedure to minimize unwanted interactions between
analytes and the acidic silanols of the silica support.
Hypersil GOLD PFP enabled the optimal separation of
all 14 analytes by improving the resolution of the earlier
eluting compounds. Hypersil GOLD PFP introduces a
fluorine group into the stationary phase to improve selec-
tivity towards halogenated compounds, as well as polar
compounds containing hydroxyl, carboxyl, nitro or other
polar groups.3
3. Separations using Acetic Acid and Trifluoroacetic Acid
(TFA) as Eluent Modifier
Trifluoroacetic acid, formic acid and acetic acid can be added
into the mobile phase to generate differences in selectivity.
Separation of 14 illicit drugs on a Hypersil GOLD PFP
column was evaluated by using either trifluoroacetic acid,
formic acid or acetic acid as eluent modifier. The separation
method with 0.02% TFA (Figure 2A) provided fast separation
performance with good resolution and sharp peaks.
However, the use of TFA is generally not recommended
with MS detection due to its effect on signal suppression.
All of the analytes are well resolved with 0.1% formic
acid as modifier (Figure 1C), but only when 100% water
is used at the beginning of the gradient method (Method C).
Prolonged use of 100% water may degrade the stationary
phase and shorten the column lifetime, so gradient method
C is not suited for routine use.
Most of the analytes are well separated with adequate
resolution using 0.06% acetic acid as eluent modifier
(Figure 2B). However, under such conditions, a few pairs
of compounds, such as oxycodone and methamphetamine
(peaks 7 & 6), hydrocodone and 3, 4-MDMA (peaks 5 & 8),
cocaine and noscapine (peaks 10 & 11), are not baseline
resolved.
4. Separations with Hybrid Column Phases
Three hybrid stationary phases were evaluated after
connecting different stationary phase columns in series:
Figure 3A: 50 x 2.1 mm Hypersil GOLD + 50 x 2.1 mm Hypersil GOLD PFP
Figure 3B: 50 x 2.1 mm Hypersil GOLD PFP + 50 x 2.1 mm Hypersil GOLD
Figure 3C: 100 x 2.1 mm Hypersil GOLD PFP + 20 x 2.1 mm Hypersil GOLD
Separations of 14 illicit drugs with these three hybrid
stationary phases demonstrated great variation in selectivity.
In general, the hybrid column phases improved selectivity
between THC and cannabinol, cocaine and noscapine, but
reduced selectivity between earlier eluting compounds,
such as oxycodone, MA, hydrocodone and MDMA,
compared with the Hypersil GOLD PFP phase.
5. Separation with Ternary Gradient
The separation of the drug mixtures was dramatically
improved by using three solvents: water, acetonitrile and
methanol (Figure 4). Baseline resolution of all 14 drugs
was achieved. Methanol, a weaker eluent compared with
acetonitrile, provided better resolution for most of the
analytes. However, the flow rate had to be reduced to
accommodate high column backpressure caused by the
high viscosity of methanol. Adding acetonitrile reduced
the column backpressure so as to maintain the same
separation speed.