Evaluation of reproducibility and sensitivity of a new hardware workflow for
LC/MS analysis of plasma samples.

Karen E. Yannell, Genevieve C. Van de Bittner, and Daniel J. Cuthbertson

Introduction

Results and Discussion

LC/MS for Targeted Metabolomics
Targeted metabolomics studies using liquid
chromatography mass spectrometry (LC/MS) are a welltested approach for interrogating known pathways. As
these are becoming more common place in researchlaboratories, the boundaries are being pushed into
making methods that are the most routine, reproducible,and analytically sensitive. This poster describes aworkflow that can achieve reproducible results throughautomation of sample preparation coupled with ananalytically sensitive analysis using a LC designed formetal sensitive analytes and an ion funnel triple
quadrupole mass spectrometer (TQ).

Experimental
Infinity II Bio LC for Metal Sensitive Analytes
After the sample was prepared, 3 µL of the reconstituted
extract was injected onto an Agilent HILIC-Z column (2.1x 150 mm) and analyzed with an ammonium acetate
buffer, pH 9. Standard LC systems need to be passivated
to remove excess metal ions which can cause peaktailing and reduce overall analytical sensitivity. The newInfinity II Bio LC has MP35N metal alloy which is much
lower in free metal ions. This system show significant
improvement and was used for this method withoutpassivation.

Metabolomics
2021

Results and Discussion

BioLC is Easy to Use and Provides Low LODs and RT
Reproducibly for Metabolites
The BioLC was working without any specialized cleaningor additives for metal sensitive analytes like adenosine
diphosphate (ADP). 50 ng/mL standard had immediatesignal after installation with the BioLC while a stainlesssteel LC had no signal (Figure 3). After passivation of thestainless-steel LC the signal was regained. This showsincrease ease of use with the BioLC for metabolomics
applications.

6495C had Reproducible Signal for the Metabolite
Extract from 10 µL Bovine Plasma
Bovine extract injections led to sensitive andreproducible metabolite detection. 3 µL injection of theplasma sample extract had a relative standard deviation(RSD) of 1-14% for the analytes measured. Examples ofthe analytes detected, and their RSDs are in Figure 5.
The HILIC Z chromatography is stable (Figure 4) and also
gives unique chromatographic separation of isobaric
analytes like iso/leucine (Figure, a).

a

RSD = 1%bRSD = 14%c

RSD = 3%d

RSD = 6%

Experimental

Figure 1: Bravo chassie contains 9 positions forsolvents, tips, plate shaker and vacuum filtration station.

Figure 2: Infinity II Bio LC stack included a multisampler,column compartment with bio compatible switchingvalve, and high speed 1300 bar pump all made frombioinert material for improved detection limits for metalsensitive analytes. The 6495C LC/TQ was used in thismethod because the ion funnel improves detectionlimits.
6495C Ion Funnel TQ for Reproducible and Low
Detection Levels
An Agilent Jet Stream (AJS) source was used to ionize
analytes. The source conditions and ion funnel wereoptimized for the lowest responders, but the ion funnel
on the 6495C (Figure 2) had a broad range of optimal
conditions for various classes of analytes. A dynamic
MRM method (dMRM) was used to collect one iontransition per metabolite. Ion transitions were in bothpositive and negative mode. Multiplexing was prioritized
to target as many analytes as possible. All retention
times were confirmed with pure standards. Dwell times
were variable but as low as 6 ms. Adding moremetabolites is possible with min dwell time of 0.5 ms.

Figure 3: Extracted ion chromatogram for ADP on a new
bio LC (top), new standard stainless-steel LC (middle)
and passivated stainless-steel standard LC.
Reproducibility of the retention times (RT) over multiple
HILIC columns was evaluated to ensure stability and thata narrow RT window can be used. Low variation wasobserved even when buffers and columns were changed
frequently for the purpose of forcing variability in theexperiment (Figure 4).
8RT (min)Automated Sample Preparation
A Metabolomics Bravo liquid handler platform (Figure 1)
was used to process 10 µL aliquots of pooled bovine
plasma. This Bravo-automated workflow has been
described previously and provides more reproducible
results than three individual technicians manually
preparing samples.1 Briefly, plasma aliquots in a 96-wellplate have their proteins precipitated and the sample istransferred to the Captiva EMR-Lipid extraction plate.
Here, the protein precipitate is captured by a filter, thelipids are captured by the EMR-Lipid resin, and the polar
metabolites pass through the plate. Wash steps are thencompleted to boost metabolite recovery. The
Metabolomics Bravo platform is equipped with a shaking
station and vacuum filtration which facilitate sample
mixing and collection of the metabolite-containing eluent.
Lipids remain on the EMR-Lipid resin and can be extractedusing a second method if desired. The metabolitecontaining sample is dried down offline and reconstituted
in the appropriate LC/MS analysis solvent. Data in thisposter was obtained from 10 µL aliquots, but theprotocols can be adjusted up to 100 µL sample.

L-Leucine7

L-Isoleucine

6L-Methionine

5L-Tyrosine

4Valine

30

100

200

300

# injections

400

L-Proline
Succinic acid

Figure 4: RT stability of various metabolites in theplasma extract over 400 injections and three different
columns.

Figure 5: Leucine and Isoleucine (a), alphahydroxyglutaric acid (b), succinate (c), and transaconitic acid (d) and the RSD of 6 injections of bovine
plasma extract.

Conclusions
The Combination of these Instruments Produce aSensitive and Reproducible Platform for Targeted
Metabolomics Studies• Bravo Metabolomics Workbench was able to extract
analytes from as little as 10 µL plasma and the extract
was well separated by the HILIC Z column• HILIC Z and the Infinity II Bio LC gave sensitive andreproducible analysis even over multiple columns.
• The 6495C Ion Funnel TQ gave sensitive andreproducible analysis when testing a complex matrix

References
1 Sartain, M., Gomez, M., Van de Bittner, G., and Shu, H. Enabling Automated,Low-Volume Plasma Metabolite Extraction with the Agilent Bravo Platform. 59942156EN. 2020.

For Research Use Only. Not for use in diagnostic procedures.
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