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INTRODUCTION

The use of data independent acquisitions in a metabolite identification workflow is commonplace

1,2; however, when all

ions are recorded, processing the data can be challenging especially in complex matrices where significant interference from unwanted background ions can cause low level metabolites to be missed. It is widely accepted that there is a correlation between an ion’s m/z ratio and its ion mobility drift time (dt) for a given class of compounds (for example lipids, peptides, oligosaccharides etc.)

3 and as such this relationship can be

exploited to remove interferences from background ions. This poster will describe the investigational use of prototype software that allows quadrupole filtering during the IM cycle of an HDMS

E experiment (referred to as Trend Directed HDMS

E)

to remove background and interfering ions thus reducing the complexity of met ID sample sets. The ions are removed based on a differing relationship of m/z and drift time to the expected metabolites and the parent drug. This results in a reduced data file size, which collectively allows for faster and more efficient data interpretation and processing. Here we also describe the use of UNIFI and the embedded workflow steps to collect, process and evaluate met ID sample sets.

SYNCHRONIZING ION MOBILITY WITH QUADRUPOLE MASS FILTERING FOR HIGHLY EFFICIENT BACKGROUND REMOVAL DURING ACQUISITION FOR METABOLITE IDENTIFICATION

Jayne Kirk1, Darren Hewitt1, Martin Palmer1, Jason Wildgoose1, Richard Denny1 and Mark Wrona2 1Waters Corporation, Wilmslow, UK, 2Waters Corporation Milford, MA, US

References

1. Bonn B. et al., Rapid Communications in Mass Spectrometry, 2010, 24, 3127-3138.

2. Blech S et al., International Journal for Ion Mobility Spectrometry, 2013, 16:1, 5–17.

3. McLean J. et al., Analytical Chemistry 2014, 86, 2107-2116.

RESULTS AND DISCUSSION

Data were initially acquired in HDMSE mode and the m/z to dt relationship for buspirone and nefazodone and their respective

metabolites was determined by viewing data in the Ion Mobility Data Viewer (Figure 3 a). The observed drift times were then

corrected for the transit times through the various lenses of the Vion IMS QTof, a m/z to dt reference table created and acquisition

under Trend Directed HDMSE preformed. Figures 3 b and c show the results for buspirone and nefazodone, respectively, with a

significant number of background and interfering ions removed through synchronisation of the IM separation and quadrupole.

Shown in Figure 4 is the HDMSE spectra of the glucuronide of the hydroxylated metabolite of buspirone along with fragment ion

assignment denoted by the blue icon. Drift time (in addition to retention time) alignment is also used for spectral clean up in the

low and high energy functions leading to unambiguous assignment of fragment ion information.

CONCLUSION

Synchronising the quadrupole with ion mobility separation can be used to effectively remove background interfering ions for the application of metabolite identification.

Trend Directed HDMSE data acquisition allows for decreased

size of data files and faster processing thus reducing the burden on data interrogation and storage.

Navigation of large, complex data sets: organization of metabolite spectra; observation of trend plots; structure elucidation in a single workflow.

Future work: Investigate the use of multiple trend lines within a single acquisition to analyse various classes of small molecules.

METHODS

INSTRUMENTATION

All data were acquired on a Waters ACQUITY UPLC I-Class coupled to a Vion IMS QTof with an electrospray source. Data were acquired in HDMS

E and Trend Directed HDMS

E modes

using prototype software. Trend Directed HDMS

E is a novel acquisition mode that

synchronises the quadrupole with the ion mobility (IM) separation and only allows the detection of ions that have a defined m/z to dt relationship. The quadrupole is operated in resolving mode and is controlled via a look up table that defines the set mass at time intervals after the IM separation starts, thereby only transmitting a particular m/z window at a given dt (Figure 1).

Separate trend lines were created for buspirone and nefazodone (Figure 2). As can be seen from the buspirone trend line (green line) the metabolites and parent follow a linear m/z to dt relationship. Whereas some of the phase II metabolites of nefazodone do not exhibit a linear trend with the parent and other metabolites. In order to maximise sensitivity of all nefazodone metabolites the m/z to dt reference table comprised of a linear trend between m/z 100 - 520 (blue line) and then jumped between static m/z values at specified drift times for the non-linear phase II metabolites (characterised by the blue squares in Figure 2). It should also be noted that it is possible to move the quadrupole both forwards and backwards within the reference table as can be observed by the sequence used for nefazodone phase II metabolites at m/z 634.2 → 678.3 → 662.3 → 678.3.

LC/MS CONDITIONS

Column: ACQUITY HSS T3 1.8 µm, 2.1 x 100 mm

Mobile phase A and B: water and acetonitrile + 0.1 % formic acid

Gradient: 2% to 60 % B over 7 in and up to 100% for a further min,

equilibrate for 2 min. Total time 10 min

Flow rate: 0.5 mL/min

Instrument: Vion IMS QTof operated in positive ion mode under

HDMSE and Trend Directed HDMS

E where data acquition was

between 50 - 700 Da using 0.1 sec scan time. MS low and high

collision energy was 6 and 20 - 50 eV (ramp), respectively.

SAMPLE PREPARATION

Nefazodone (10 µM) was incubated with cryopreserved rat

hepatocytes and relevant cofactors at 37◦C for 0, 15, 30, 60, 120,

and 240 minutes. The incubations were terminated by addition of

an equal volume of ice cold acetonitrile, centrifuged, and the

supernatant submitted for analysis. A 1 in 10 dilution was then

prepared in urine. Prior to its use the urine was diluted 1 in 5.

Figure 4. HDMSE spectra of the glucuronide of the hydroxylated metabolite of buspirone showing spectral clean up with drift time alignment on (right)

and off (left).

Figure 6. Comparison of the high energy spectra of nefazodone

(bottom) and the hydroxylated metabolite (top) for biotransformation lo-

calisation. The most likely point of metabolism is indicated by the bright

green colour. Here, it is suggesting hydroxylation of the aliphatic moiety.

Figure 8. Component table summarising the identified metabolites of nefazodone in the t= 60 min incubation along with accurate mass measurement ,

CCS and discovery settings such as identification of a chlorine isotopic pattern, common fragment ions and glucuronide neutral losses.

Figure 7. The

trend plot of buspi-

rone and its identi-

fied metabolites

with response plot-

ted over the ex-

perimental time

course. Hydroxy-

lated buspirone is

identified as one of

the major metabo-

lites.

Figure 1. Schematic of the VIon IMS QTof mass spectrometer. The

quadrupole transmission window changes during the IM separation, so

for a given drift time only a certain m/z region will be transmitted through

the quadrupole. This synchronisation is optimised for the analytes of

interest.

Table 1. The size of sample set is reduced by two thirds and the proc-

essing time by more than a half by using Trend Directed HDMSE over

HDMSE.

Complete Sample Set % File Size Reduction

% Processing time Reduction

Buspirone Trend Directed HDMSE 68.9 52.8

Nefazodone Trend Directed HDMSE 66.0 55.9

Figure 5. XICs of the glu-

curonide of the hydroxy-

lated nefazodone me-

tabolite are shown under

HDMSE (top) and Trend

Directed HDMSE

(bottom)

acquisition modes. The

latter shows an improve-

ment in the S/N ratio as

more noise is removed

when acquiring with

Trend Directed enabled.

The improvement in the reduction of noise from these data when using Trend Directed HDMSE is shown in Figure 5. The extracted

ion chromatograms (XIC) of the glucuronide of the hydroxylated nefazodone metabolite under HDMSE and Trend Directed HDMS

E

were measured with S/N ratios of 96 and 263, respectively.

The impact on processing time and on file size is summarised within Table 1, with the file size reduced by a third and the

processing time falling by over a half under Trend Directed HDMSE.

Data can then be interrogated using the tools and workflow steps within UNIFI for metabolite identification. The biotransformation

tool compares the high energy fragmentation spectra of the parent to the metabolite. By comparing fragment ions that are retained

or shifted in mass the algorithm highlights the parent structure with the likely site of transformation (Figure 6).

The trend plot shown in Figure 7, where the response is on the x axis and the sample on the y axis, tracks the decrease in parent

during the time course and the increase in metabolites formed. Here the main metabolite for buspirone was identified as a

hydroxylation. This plot can also be useful when eliminating false positive as their response will be intermittent or constant over

the time course.

The discovery workflow shown in Figure 8 allows further

questions to be asked of the data. Here metabolites that have

common fragment ions, chlorine isotopic patterns and neutral

losses are highlighted. This can also be useful when there are

unexpected or unknown metabolites present within the

sample set.

The quadrupole was typically scanned at a rate of

~77 kDa/s; this far exceeds the typical scan speeds of most

commercial quadrupole systems (~10 kDa/s). This elevated

scan rate would result in significant degradation of the peak

shape, however, as the quadrupole was operated in low

resolution mode (circa 60Da window), this effect was not

observed.

Figure 2- Trend Lines for nefazodone, buspirone and their respective

metabolites.

Nefazodone phase II

metabolites Linear trends for

both buspirone and nefazodone

Figure 3. Mobility viewer showing m/z vs dt for the buspirone 120 min incubation acquired in HDMSE mode with no reference table (a), with Trend Di-

rected HDMSE (b) and nefazodone with Trend Directed HDMS

E (c).

a b c = metabolite

Background removal region

Background removal region