Discovery of multiple sites of intra-molecular protonation

Discovery of multiple sites of intra-molecular protonation and
different fragmentation patterns within the fluoroquinolones
using ion mobility mass spectrometry
Sara Stead*1; Michael McCullagh1; Davor Turkovic2
1
Waters Corporation, Atlas Park, Simonsway, Manchester M22 5PP, UK ; 2 Waters GmbH, Helfmann-Park 10, 65760 Eschborn, Germany
*Corresponding author: sara _ [email protected]
RESULTS & DISCUSSION
INTRODUCTION
Fluoroquinolone (FQ) antibiotics have been administered to
livestock for different purposes, (a) prevention and control
of infections and (b) growth promotion. The use of antibiotic
growth promoting agents in animal husbandry has been
forbidden in the EU since 2006.
Tandem quadrupole mass spectrometry has gained
widespread acceptance for quantitative analysis in terms of
selectivity and sensitivity. The selection of the MRM
transitions is critical and must be performed and validated in
accordance with CD 2002/657/EC.
Ciprofloxacin elutes at retention time 2.19 mins using the
gradient conditions described. Figure 2 shows the BPI
chromatogram and the component plot summary for 9 FQ
compounds. If the data is reviewed using the component drift
plot summary, 18 mobility resolved FQ species are observed
(Figure 3). Each FQ compound was found to be comprised of
two protomers, i.e. protonation occurring at either the acidic
or basic region of the molecule. Estimated CCS values of 108.7
Ǻ2 and 119.1 Ǻ2 were determined for ciprofloxacin protomers
(Figure 4).
In this study, High Definition Mass Spectrometry (HDMS) is
explored as a tool for method development to support the
unequivocal identification of FQ residues in complex
matrices. HDMS uses a combination of high resolution MS
and high efficiency ion mobility based measurements and
separations (Figure 1). Compounds can be differentiated
based on size, shape and charge. In addition, both precursor
ion and fragment ion information can be acquired in a single
injection in an HDMS experiment (HDMSE).
Figure 5. Ciprofloxacin acid and basic group protomer MSE fragmentation
spectra generated using an IMS screening workflow in UNIFI.
Ion mobility differentiates molecules as they tumble through
a buffer gas and their progress is related to the average
rotational collision cross section (CCS). Ion mobility drift
time can be used to determine the CCS of a molecule.
Figure 2. UPLC HDMSE precursor base peak ion chromatogram for a solvent
standard containing 25 antibiotic compounds (top) and the component plot
summary view (bottom) for the 9 fluoroquinolone compounds detected
between 2.0 - 2.6 mins.
Figure 6. Illustration of identification of two protomers of danofloxacin
identified in porcine muscle extract without ion mobility spectral clean up
Figure 1. A schematic representation of a SYNAPT G2-S HDMS and
illustration of the mechanism of travelling wave ion mobility separations.
METHODS
Ciprofloxacin; enrofloxacin; danofloxacin; sarafloxacin;
norfloxacin; marbofloxacin; enoxacin; perfloxacin and
ofloxacin
Sample Preparation
The extracts of blank and fortified porcine muscle tissue
were kindly provided by RnAssays for the purpose of this
study.
MS Conditions
MS system:
Waters SYNAPT® G2-S HDMS
Ionization mode:
ESI+
Acquisition Mode:
HDMSE
Mass range:
m/z 50 – 1200
Acquisition Rate:
4 spectra/s
Capillary voltage:
2.0 kV
Cone Voltage:
25 V
Source temp:
120oC
Desolvation temp:
550oC
Drift Gases:
N2
E
Figure 3. Component plot summary for the 9 identified fluoroquinolones and
the observation of 18 ion mobility resolved (0.5 ms) species in solvent.
Figure 7. Illustration of the identification of two protomers of danofloxacin
in spiked porcine muscle extract with ion mobility spectral clean up
MS High energy:
15.0 — 45.0 V (ramped)
IMS Wave Velocity:
900 m/s
IMS Wave Height:
40 V
Resolution:
20000 (FWHM)
This data confirms that further consideration should be
given to method development since the ratio and
formation of the protomers was seen to vary with eluent
flow rate, capillary voltage, cone voltage and extract
composition. The extent of the protonation multiplicity and
CONCLUSIONS
UPLC Conditions
LC system:
ACQUITY UPLC® System
Column:
ACQUITY BEH C18, 1.7 µM,
2.1 x 50 mm, @ 45oC
Mobile phase A:
Water (0.1% formic acid)
Mobile phase B:
Acetonitrile (0.1% formic acid)
Gradient Table:
Injection Volume:10µL
Gradient: Time (min) Flow Rate
%A
%B
Initial
0.600
95.0
5.0
1.00
0.600
95.0
5.0
8.00
0.600
5.0
95.0
9.00
0.600
95.0
5.0
Data Acquisition and Processing Software
Data were acquired using MassLynx and processed using
UNIFI v 1.6.5 Research Edition software.
FQ solvent
standard mixtures were used to generate a scientific library
containing retention times, fragment ion elemental
compositions and CCS values. This permitted non-targeted
data acquisition with a targeted screen to be performed
using HDMSE data.
Figure 4. Intensity vs drift time for the protomers of ciprofloxacin with the
hypothesised respective sites of acid/basic group protonation highlighted and
the observed CCS values.
The fragments at m/z 288 & 245 (Figure 5) are believed to
arise from a species where ionisation occurs on the basic
group. Protonation occurring on the acidic group results in the
formation of fragment ions at m/z 314 & 231.
A series of porcine extracts were screened for the presence of
fluoroquinolones using the parameters optimised in solvent
standard. Figures 6 & 7 show the identification of two
protomers of danofloxacin (with and without the application of
ion mobility spectral clean-up). In this example, ion mobility
has been used to resolve the matrix derived interferences
from the identified component of interest, danofloxacin.
It can also be seen that for both protomers the following
performance was obtained; mass accuracy < 1ppm and %CCS
error is within 2% of the expected values. In addition, the
individual protomer precursor ion/fragmentation spectra were
generated.
Based on the observations of the characteristic
ionisation for the fluoroquinolone compounds included
in this study the use of UPLC HDMSE for method
development purposes is warranted.
The elucidation of different sites of intra-molecular
protonated species has been shown using ion mobility
MS and have been determined from the different
single component fragmentation spectra.
Single component precursor ion MS and MSE
fragmentation spectra are generated for all
components simultaneously.
The HDMSE observations have the potential to explain
the differences sometimes observed in interlaboratory studies where participants report results
obtained from monitoring specific MRM transitions.
CCS values can provide addition confidence along
with retention time, precursor ion accurate mass and
accurate mass fragmentation spectra in non-targeted
screening experiments.
Ion mobility separations can be effectively utilised to
resolve analyte peaks from matrix interferences and
reduce the need for complex sample clean-up and
chromatographic separations.
©2010 Waters Corporation
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