A Targeted LC-MS/MS Method and Acquisition Database Optimized forCentral Carbon Pathway Metabolites
Mark Sartain1, Amy Caudy2, and Adam Rosebrock2
1 Agilent Technologies, Inc., Santa Clara, CA, USA;

2 Donnelly Centre for Cellular and Biomolecular Research and Department of Molecular Genetics, University of Toronto, Toronto ON, Canada

Introduction

Results and Discussion

A major challenge in metabolomics is achieving reproducible,
robust chromatographic resolution with a single analytical
LC/MS method for endogenous cellular metabolites due totheir diversity of physiochemical properties.
A moremanageable chromatographic solution is to develop pathwaytargeted methods. We developed a highly reproducible androbust ion-pair based reverse phase (IP-RP) chromatographic
method enabling simultaneous analysis of >200 endogenous
cellular metabolites, along with a curated database ofretention times and optimal targeted MS acquisition
parameters based on chemical standards. The Agilent
dMRM Database and Method enables researchers to easily
implement an already-optimized method for targeted
metabolomics thereby boosting laboratory productivity.

ExperimentalSample Preparation

Results and Discussion

The Agilent dMRM Database

Data Review

The Agilent Metabolomics dMRM Database provides
optimized instrument operating parameters for the targeted
MS acquisition of >200 metabolites. The choice of precursor
and product ion ions, RTs, and RT windows were based on
extensive analysis of authentic chemical standards.
MassHunter (MH) Optimizer Software was used to determine
optimal collision energies and fragmentor voltages for eachmetabolite to maximize sensitivity. The combination of thesecurated, optimized parameters generates results of thehighest quality.

With large multi-compound sample batches typically acquiredwith the Metabolomics dMRM method, MH Quantitative
Analysis software provides easy and quick method creation
for data review and peak integration. The batch type format
offers helpful features such as compound-at-a-glance data
review (Fig. 5). Reports can be automatically generated
within the software and serve as input for the next phase ofstatistical analysis.

Compound transitions are directly imported into MH
Acquisition software from the Database Browser to easily
build tailored dMRM methods (Fig. 2). Users can also
optimize new compounds of interest with MH Optimizer andresults are automatically saved to the database.

More than 200 individual authentic chemical standards were analyzed
for database curation. Cultures of Saccharomyces cerevisiae (s288cbackground) were grown in synthetic media containing either acetate
or glucose (2% w/v) as the sole carbon source. Cultures were grown tomid-log phase (OD600=0.4), vacuum filtered onto 0.2μm nylon
membranes, and immediately immersed in -20˚C ACN:MeOH:H2O(40:40:20) for quenching and extraction. Clarified metabolite extracts
were dried under nitrogen and resuspended in water prior to LC-MSanalysis.

UHPLC-MS/MS Method

The IP-RP method employs a UHPLC C18 column with the ion-pairing
agent tributylamine (TBA) added into the LC mobile phases. TheLC/MS method leverages the 1290 Infinity II UHPLC system coupled toa 6470 Triple Quadrupole LC/MS system equipped with a Jet Stream
electrospray ionization source (Fig. 1), operated in dynamic multiple
reaction monitoring (dMRM) mode with negative ion polarity. The
method also implements a phase-matched guard column and switchingvalve to backflush the guard and analytical column during each run.
This combination effectively clears matrix contaminants for addedrobustness. Major UHPLC parameters are as follows:
Agilent UHPLC 1290 Infinity II Systemwith the 1290 Infinity II Flexible Pump (Quaternary Pump)
ColumnAgilent ZORBAX Extend C18, RRHD,
2.1x150mm 1.8 um (p/n 759700-902)

Guard Column

Agilent ZORBAX Extend C18 Fast Guard
2.1 x 5mM, 1.8 um (p/n 821725-907)

Column Temperature

35 ºC

Gradient, Flow Rate(Analytical Portion)

Time (min)0%13.055%

--

45%

0%

20.01%--

99%0%

24.01%--

99%0%

2002468103

12

Second Laboratory Observed RT (min)1416> 60 sec

20%

145 to 60 sec--

430 to 45 sec

80%

16

10

15 to 30 sec

7.5•

A robust IP-RP chromatography optimized for challenging
ionic metabolites and suitable for analyses in complex
cellular matrices•

Compound-specific optimized dMRM MS acquisition
parameters•

Pre-defined compound retention times and retention timewindows•

A customizable database format

69 to 15 sec

0%3783 to 9 sec

0%

10-3 to +3 sec--The combined UHPLC-MS/MS method and acquisition
database provides:

12-9 to -3 sec

100%Conclusions

14-15 to -9 sec

2.5Figure 6. Pathway Architect view of TCA Cycle (KEGG). Matchingcompounds are highlighted, and heat strips indicate normalizedabundances.

16-30 to -15 sec

DTo demonstrate the workflow, metabolite extracts from yeast
cells cultured with acetate or glucose as the sole carbon
source were analyzed with the full dMRM method. After
processing the datasets with MH Quant and MPP, pathway
analysis indicated several affected pathways including theTCA cycle (Fig. 6). Notably, pyruvate was found to be
reduced in the acetate-grown cells, likely due to the lack of
glucose as a substrate for glycolysis.

18-45 to -30 sec

C40.0 minThe IP-RP method is fully detailed and designed to be easily
replicated across laboratories with little observed variationfrom the DB compound RTs, enabling narrow dMRM RT
windows and providing added confidence in assigning MRM
signals to the correct metabolite in a complex matrix (Fig. 4).

-60 to -45 sec

B

To enable multivariate relative comparisons Agilent’s Mass
Profiler Professional software (MPP) provides the ability to:• Easily import, compare, and visualize LC/MS data
• Find meaningful relationships in complex experiments• Identify affected biological pathways with the Pathway
Architect module

Figure 3. Overlapped MRM chromatograms of more than 100
metabolite standards at 5 ng on-column.

< -60 secA… backflushing and regeneration phase
Total Run Time

An ion-pair based reverse phase (IP-RP) chromatographic
method was developed that leverages the proven robustnessof Agilent C18 UHPLC columns, with added functionalizationof the stationary phase with the ion-pairing agent
tributylamine to facilitate retention of polar metabolites frommultiple functional classes (Fig 3.). The presence of the ionpairing ternary amine TBA in the chromatography was foundto facilitate retention of acidic metabolites, while non-acidic
metabolites still maintained conventional reverse-phaseselectivity.

Curating Laboratory RT (min)0.25 ml/min

The Agilent dMRM Method

Retention Time Agreement

A= 97:3 Water/Methanol with 10mM
Tributylamine, 15mM Acetic Acid
B = Isopropanol (not used)
C= Methanol with 10mM Tributylamine,
15mM Acetic Acid
D = Acetonitrile (backflushing solvent)Analytical Flow Rate

Differential Analysis and Pathway Mapping
Figure 2. Database Browser view of Metabolomics dMRM Database

Figure 1. The Agilent Infinity II UHPLC
System with the Agilent 6470 Triple
Quadrupole Mass Spectrometer

Mobile Phase

Figure 5. Compounds-at-a-glance view within Quantitative Analysis
software, showing 5 compounds (columns) across 3 samples (rows).

18Figure 4. To demonstrate method consistency retention times fromthe curating laboratory were verified at a second laboratory with asubset of standards (n=67). Excellent agreement was observed withan average  RT <0.1 minute. The insets shows a histogram of thecompound RT deviations observed between the two laboratories.

For metabolomics researchers needing targeted methods
with many metabolites per method, the database will allow
fast startup and provide excellent results without the user
needing to optimize each and every compound.

For Research Use Only. Not for use in diagnostic procedures. This information is subject to change without notice. RA44448.4276041667