Application Note
Metabolomics

Enabling Automated, Low-Volume
Plasma Metabolite Extraction with theAgilent Bravo PlatformAuthors
Mark Sartain, Manuel Gomez,
Genevieve Van de Bittner, andHenry Shu
Agilent Technologies, Inc.

Abstract
This application note describes the modification of the Agilent Bravo Metabolomics
Sample Prep Platform to lower the required starting plasma volume from 100
to 25 µL. Analyte recovery of the low-volume method was assessed using anAgilent 6546 LC/Q-TOF, and reproducibility was compared with manual sample
preparation. The method was shown to enable outstanding metabolite recovery andsuperior reproducibility compared with manual sample processing methods.

Introduction

Experimental

In basic and translational researchsettings, sample preparation before
LC/MS-based analysis of plasma
metabolites is challenging for several
reasons, including the presence ofcompounds with different physicalproperties, variability between operators,
and interday reproducibility. Additionally,in some research settings, limited
amounts of plasma can be obtained
from infants/children or from animalmodels. Here is a description of amodification to the Bravo Metabolomics
Sample Prep Platform to accommodate
25 µL of plasma sample volume. Like theoriginal protocol, this modified method
precipitates plasma proteins to quench
enzymatic activity, depletes lipids, andextracts metabolites, providing a clean
metabolite sample for LC/MS analysis.
An Agilent 6546 LC/Q-TOF was used
to evaluate metabolite recovery of theautomated 25 µL method and also
compared the reproducibility against
manually prepared samples processedby multiple laboratory staff.

Materials

2Normal human plasma (lithium heparin,pooled, mixed gender) was procured
from BioIVT (Hicksville, NY, USA). TheMass Spectrometry Metabolite Library(MSMLS) (IROA Technologies) waspurchased through Millipore Sigma(St. Louis, MO, USA). Unlabeled yeast
metabolite extract “ISO1-UNL” and U-13C
labeled yeast metabolite extract “ISO1”(ISOtopic Solutions) were purchased
through Cambridge Isotope Laboratories,Inc. (Tewksbury, MA, USA).
Agilent LC/MS-grade acetonitrile(part number G2453-85050) was usedfor LC/MS analysis. Ultrapure water wasproduced with a Milli-Q Integral systemequipped with a LC-Pak Polisher and a
0.22 µm point‑of‑use membrane filter
cartridge (Millipore Sigma). LC/MS grade
ammonium acetate was purchasedfrom Millipore Sigma. To minimize thebinding of polar ionic metabolites totrace levels of metal in the LC system,
Agilent InfinityLab deactivator additive(part number 5191-4506) was added tothe mobile phases.

Instrumentation•

Agilent Bravo Metabolomics
Sample Prep Platform(part number G5589AA)•

Agilent PlateLoc thermal microplate
sealer (part number G5585BA)•

Agilent 1260 Infinity II Prime LC
including:• Agilent 1260 Infinity II Flexible
Pump (G7104C)• Agilent 1260 Infinity II
Multisampler with thermostat(G7167A)• Agilent 1260 Infinity II
Multicolumn Thermostat(G7116A)•

Agilent 1200 Isocratic Pump
(G1310A) with a 100:1 splitter(G1607-60000) for reference massaddition•

Agilent 6546 LC/Q‑TOF with a Dual
Agilent Jet Stream technology ion
sourceBravo consumables•

Agilent Captiva EMR—Lipid plates(part number 5190-1001)•

Agilent 250 μL disposable tips
(part number 19477-002)•Agilent PlateLoc peelable aluminumseals (part number 24210-001)•Agilent Captiva collection plates(part number A696001000)•

Agilent reservoirs, single cavity(part number 201254-100)•

Thermo Scientific Matrix 1.0 mLscrew top tubes and rack, 2D
V-bottom (Thermo Scientific,part number 3741)

Bravo method
Beginning with the Bravo Metabolomics
Sample Prep Platform (p/n G5589AA)1(original method), modifications weremade to the protocol that include
reduction of the starting plasma volume
from 100 to 25 µL. Figure 1 summarizes
major steps, and Figure 2A is adisplay of the custom application userinterface used for low-volume plasma
metabolite extraction.
Many of the steps and fundamental
principles between the original method
and this modified protocol remained
the same. Importantly, these included
maintaining a 225:225:100 ratio of
methanol:ethanol:plasma for effective
protein precipitation and use of Captiva
EMR—Lipid plates for protein filtering andlipid depletion, as described previously.1
In addition, this protocol included
a 10-minute incubation following
quenching, subsequent water addition
to the quenched sample to achieve 50%
final water content, and washing theCaptiva EMR—Lipid plate twice with 1:1:2
methanol:ethanol:water. These combined
measures improved recovery for a small
percentage of metabolites (data notshown). With the original method, ~55%of the metabolite extract is transferred
to a final plate for dry down. In contrast,
the entire combined volume of Captiva
EMR—Lipid eluant including washes
is collected and dried in the modified
low-volume plasma protocol, thereby
minimizing the overall losses otherwise
expected when moving from 100 to
25 µL plasma input.

Low volume plasma protocol onsite

Place plasma, 25 µL per tube, in a 96-tube rack.

Transfer 112.5 µL of 1:1 methanol:ethanol to plasma,
pipet mix and shake; wait 10 minutes.

Transfer 87.5 µL of water to quenched plasma,
pipet mix and shake; wait 10 minutes.

Transfer the sample to an Agilent Captiva EMR—Lipid plate.

Remove proteins and lipids; collect metabolites in the filtrate.

Wash the Captiva EMR—Lipid plate twice with 250 µL of 2:1:1
water:methanol:ethanol; collect metabolites in filtrate.

Dry the samples (optionally store).

Reconstitute the samples in 100 µL of a suitable LC/MS solvent.

LC/MS Analysis
Figure 1. Workflow for onsite, automated, low-volume metabolite
extraction from plasma. Green steps are performed with theAgilent Bravo platform.

3

AB

Figure 2. Custom Agilent Bravo settings used for low-volume plasma metabolite extraction (A) and reconstitution (B).

4The original method offers both an“offsite” and “onsite” version, wheremetabolite quenching is either done at
a separate location or automated on
the Bravo platform, respectively. The
only evaluation was an onsite versionof this modified low-volume protocol,
though an offsite version that simply
includes a subset of automation steps
from the onsite version could easily beaccommodated.
For sample reconstitution, the same
application settings were used as theoriginal method, except modifications
were made to specifically optimize
for downstream sample injection andanalysis with HILIC chromatography(Figure 2B). Eighty microliters ofacetonitrile (in lieu of 20 µL of methanol)
was added to the dried samples, shaken,
and followed by addition of 20 µL (inlieu of 80 µL) of water and shaken, still
resulting in 100 µL final volume for thereconstituted samples. Plates weresealed immediately with PlateLoc toprevent evaporation.

LC/MS method
Experimental LC/MS methods werelargely followed as previously described,2except the LC gradient was adjusted tocompensate for the larger delay volumeof the quaternary pump used in thisstudy. Detailed chromatography andMS parameters are provided in Tables 1and 2.

Chromatographic conditions and MS parameters
Table 1. Agilent 1260 Infinity II Prime LC conditions.
Parameter

Value

Analytical Column

Agilent InfinityLab Poroshell 120 HILIC-Z, 2.1 mm × 150 mm, 2.7 μm, PEEK-lined(p/n 673775 924)

Column Temperature

25 °C

Injection Volume

10 µL

Autosampler Temperature

4 °C

Needle Wash

6 seconds in wash port (50:50 water/methanol)

Mobile PhaseA) 10 mM ammonium acetate in water with 2.5 µM Agilent InfinityLab deactivator
additive, pH = 9B) 10 mM ammonium acetate in water/acetonitrile 15:85 (v:v) with 2.5 μM InfinityLabdeactivator additive, pH = 9Flow Rate

0.25 mL/min

Gradient Program

Time
0.00
1.00
4.50
7.50
8.00
13.00
16.00
22.00
23.00
23.50
25.00

Stop Time

25.00 minPost Time

3.00 minObserved Column Pressure

170 to 330 bar%B9696888886868265659696

Table 2. Agilent 6546 LC/Q-TOF conditions.
Parameter

Value

Ionization Polarity

Negative

Ionization Source

Dual AJS

Gas Temperature

225 °C

Gas Flow

13 L/min

Nebulizer

35 psig

Sheath Gas Temperature

350 °C

Sheath Gas Flow

12 L/minVCap

3,500 V

Nozzle Voltage

0V

Fragmentor

125 V

Skimmer

45 V

OctopoleRF Vpp

750 V

Reference Massm/z 59.01385, m/z 980.016375 (–)

MS Rangem/z 50 to 1,600

MS Acquisition Rate

1.5 spectra/s5Software
Agilent VWorks (Ver 13.1.4) automation
control software was used to control
the Bravo system. Agilent MassHunter
Q-TOF Data Acquisition version
10.1 was used to operate the 6546
LC/Q-TOF. Agilent MassHunter PCDLManager version B.08 SP1 was used tomanage and edit the subset libraries,
created from a subset of the Agilent
MassHunter METLIN PCDL version
B.08. MS data were quantitated using
Agilent MassHunter Quantitative
Analysis software (version 10.1), andthe quantitative method was createdfrom the custom PCD (with curated RTinformation) containing the metabolite
targets. All quantifiers were based on the[M-H]– ion.

Assessment of automated versus
manual protocol performance
Sixty 25-µL plasma samples wereprocessed with the low-volume plasma
protocol using the Bravo instrument.
A manual version of the protocol
was provided to three experienced
technicians and each processed
20 samples. The manual protocol wasdesigned to emulate the experimental
steps of the Bravo protocol as closely
as possible. Figure 3 illustrates theexperimental workflow. Except for pipettetips, the same consumables were used.
Hand pipettors, a benchtop vacuumfiltration station, and a thermal mixer
were used in lieu of the Bravo instrument
and accessories. The same starting
pooled plasma aliquot and the same
reagent bottles were used (same lotnumbers) for both the Bravo and manualexperiments, and all experiments wereconducted on the same day.

25 µL Aliquots (n = 120) froma single pooled plasma sample

Recovery estimation
One vial of ISO1 U-13C yeast
extract (~2 × 109 Pichia pastoris
cells) was dissolved in 400 µL of
1:1 methanol:ethanol (M:E), andinsoluble particulate was removed bycentrifugation. For the set of six prespikesamples, 10 µL of ISO1 was added to
25 µL of thawed plasma (hand-pipetted).
For the set of six postspike samples,
10 µL of 1:1 M:E was hand-pipetted to
25 µL of thawed plasma. The remainderof the low-volume automated protocol
was the same, except 102.5 µL insteadof 112.5 µL of 1:1 M:E was used forquenching, thereby ensuring a consistentratio of methanol:ethanol:plasma forall experiments. Following Captiva
Lipid—EMR collection, 10 µL of 1:1 M:E
and 10 µL of ISO1 were added to the preand postspike sample wells, respectively.
The plate was dried by vacuum
concentration and reconstituted asdescribed. The resulting 13C-compoundpeak area ratios from six pairs of preand postspiked samples were used to
calculate metabolite recoveries and %RSD values.

Metabolite extraction
Manual protocol
3 Users (n = 20 each, 60 total)

Metabolite extraction
Automated protocol
Bravo (n = 60)

U-13C Labeled
metabolite addition

Dry down

×3

Dry down

Reconstitution
with Bravo

Agilent 6546 LC/Q-TOF
Randomize injection sequence

Figure 3. Workflow strategy for automation versus manual metabolite-extraction comparison.

6To remove any effects of LC/MS
instrument variation during data analysis,
ISO1 U-13C yeast extract was added toall plasma extracts following both theBravo and manual protocols. Briefly,
one vial of ISO1 yeast extract (~2 × 109
Pichia pastoris cells) was dissolvedin 2 mL of water, and 8 µL was added
to each sample well before drying
and reconstitution. All samples werereconstituted with the Bravo protocol
in preparation for HILIC-LC/Q-TOF
analysis. The sample injection order wasrandomized.
Normalized peak areas werecalculated according to Equation 1.

Results and discussion
Tiered selection of targets provides
confident metabolite IDs
From the untargeted LC/Q-TOF
datasets, lists of confident metabolite
identifications were generated, with
their selection based on an unbiased
selection approach to obtain arepresentative understanding of themodified Bravo protocol performanceacross chemical classes (Figure 4).
Selecting potential metabolites in plasma
limited the experiment to the presenceof corresponding metabolites in theISO1 13C-labeled yeast extract used forrecovery estimation and normalization
purposes. It was additionally limited tothe most abundant yeast metabolites,
because a relatively low amount ofISO1 was intentionally added for spiking
purposes to minimize confounding
variables from the yeast matrix.
Leveraging a chemical standard library(MSMLS), both MS/MS and retention
time information were used to build
curated subset PCDs, which were used
to build methods for the MassHunter
Quantitative Analysis software.

Peak area of endogenous 12C plasma metabolite
Peak area of corresponding 13C yeast metabolite

× Average of corresponding 13C metabolite peak areas
across 120 samples

Equation 1.

List from manufacturer of common metabolites found in
unlabeled yeast extract (ISO1-UNL)
Observe single clear LC/MS peak from MSMLS?
Observe single clear LC/MS peak in ISO1-UNL
corresponding to MSMLS RT?
Same MS/MS spectra for MSMLS and ISO1-UNL?

Observe single clear 13C signal at same RT in ISO1?

Recovery experiment,n = 31

Observe single clear endogenous 12C signal in plasma
at same RT with same MS/MS spectra in abundance?

Auto versus manual
reproducibility
experiment, n = 18

Figure 4. Metabolite selection strategy.

7

The low-volume method
provides overall excellent
metabolite recoveries

C Fumaric acid (-EIC 119.0171)

132.5

Recovery
93.6%

1.5

64.7%

1.5

1.0

1.0

0.5

0.50

Recovery

2.0Counts

2.0Counts

C L-Arginine (-EIC 179.1245)

×105

2.5

0
9.5

10.0

10.5

11.0

15.0

15.5

Acquisition time (min)

16.5

17.0

30262520151045
100%R <500%R 50 to 80

Figure 6. Histogram summary of recoveries.

816.017.5

18.0

Acquisition time (min)Figure 5. Example EICs for two selected metabolites across six prespike samples (red) and six postspike
samples (black).

Number of compounds

The ISO1 U-13C labeled yeast extract
was spiked into plasma before andafter low-volume Bravo metabolite
extraction, and the 13C metabolite peakarea ratios (prespike/postspike) wereused to estimate metabolite recoveries.
Figure 5 shows chromatograms fortwo metabolites. Figure 6 shows ahistogram summarizing the recoveries,and Table 3 lists recoveries for eachmetabolite. Excellent recoveries(>80%) were observed for 26 of the31 compounds covering several
chemical classes. The compound
D-fructose 1,6-bisphosphate showedpoor recovery (38.7%), however,this compound was considerednonendogenous to plasma as it was notfound at detectable levels.

13×105%R 80 to 120%R >120

Table 3. Metabolite recoveries for identified metabolites.
Endogenous toPlasma

Spiked 13C Metabolite

METLIN

CAS% Recovery% RSD

Amino Acids and Derivatives
Glycine2056-40-6

91.6%

4.6%

L-Alanine

1156-41-7

89.2%

1.6%

L-Arginine

1374-79-3

64.7%

2.4%

L-Asparagine

1470-47-3

87.2%

8.4%

L-Aspartic acid1556-84-8

91.6%

5.6%

L-Citrulline

16372-75-8

88.1%

3.0%

L-Glutamic acid

1956-86-0

92.5%

5.1%

L-Glutamine

1856-85-9

91.0%

3.3%

L-Histidine

2171-00-1

90.0%

6.3%

L-Isoleucine

23

73-32-5

84.4%

9.2%

L-Leucine2461-90-5

84.8%

6.2%

L-Proline

29

147-85-3

90.1%

4.8%

L-Serine30

56-45-1

96.1%

7.8%

L-Threonine

32

72-19-5

91.1%

1.8%

L-Tryptophan

33

73-22-3

94.6%

8.1%

L-Tyrosine

34

60-18-484.5%

6.1%

L-Valine

3572-18-4

78.6%

8.9%

296

979-92-0

90.3%

8.6%

SAH/S-adenosyl-L-homocysteine

Nucleobases, Nucleosides, and Nucleotides
Adenine85

73-24-5

77.1%

13.2%

5'-AMP / adenosine5'-monophosphate

34478

61-19-8

89.9%

18.1%

IMP / inosine 5′-monophosphate

3490

131-99-7

84.3%

16.4%

90

58-96-8

101.2%

10.3%

alpha-Ketoglutaric acid119328-50-7

95.5%

10.2%

Fumaric acid3242

110-17-8

93.6%

6.9%

D-Gluconic acid345

526-95-4

92.7%

6.8%

Malic acid118

6915-15-7

84.7%9.1%

UridineOrganic Acids

Sugars, Sugar Alcohols, and Sugar Phosphates
D-Arabitol

63139

488-82-4

92.3%

2.5%

D-Fructose 1,6-bisphosphate147488-69-7

38.7%

11.9%

D-Mannose 6-phosphate

5987

3672-15-9

72.8%

12.7%

Trehalose3479

99-20-7

84.1%

9.4%

53-84-9

81.3%

7.3%

Average

85.9%

7.7%

Vitamins and Coenzymes
Nicotinamide adenine
dinucleotide (NAD)101

9

Automation improves reproducibility

10

Automated Preparation

Manual Preparation

1E+6
L-Asparagine

User 1
RSD = 5.6%

Bravo
RSD = 3.5%

1E+6Normalized peak area

The interday reproducibility of theautomated original method waspreviously demonstrated to be better
compared to manual preparation.1 A
different but complementary approachto assess potential improvements inreproducibility offered by automation inlaboratory environments where there areoften multiple technicians was taken forthis application note. The reproducibilityof the low-volume plasma Bravo protocol
was compared to a manual protocol
with three different users. Figure 7
shows results for two representative
metabolites, and Table 4 summarizes theresults for all metabolites. Overall, Bravo
metabolite extraction reproducibility wascomparable to User 2, and outperformed
Users 1 and 3. For all metabolites, theBravo % RSDs were significantly lower
than the combined % RSDs for the60 manually prepared samples acrossthe three users.

Metabolite response across samples

User 3
RSD = 8.9%

User 2
RSD = 4.4%

8E+56E+5

alpha -Ketoglutaricuacid

4E+50E+0

User 1
RSD = 6.8%

Bravo
RSD = 5.9%

2E+50204060

User 3
RSD = 8.9%

User 2
RSD = 4.3%

80120

100Sample numberFigure 7. Normalized peak area % RSDs for L-asparagine and alpha-ketoglutaric acid (the actual injection
order was randomized).
Table 4. Normalized peak area % RSDs across metabolites.

Metabolite

Bravo
n = 60User 1n = 20User 2n = 20User 3n = 20Users
Combinedn = 60α-Ketoglutaric acid

5.9%

6.8%

4.3%

8.9%

10.2%

Fumaric acid

7.5%

5.2%

7.0%

9.8%

10.3%

Glycine

4.1%

6.2%

5.2%

7.3%

9.1%

L-Alanine

5.5%

8.0%

4.9%

9.8%

11.4%

L-Arginine

6.0%

7.6%5.7%

13.8%

12.6%

L-Asparagine

3.5%

5.6%

4.4%

8.9%

9.9%

L-Aspartic acid

3.9%

6.2%

4.7%

9.4%

9.7%

L-Citrulline

3.1%

5.5%

2.6%

8.5%

9.4%

L-Glutamic acid

3.3%

6.4%

2.8%

9.4%

10.3%

L-Glutamine

3.6%

5.1%

2.8%

9.4%

10.1%

L-Histidine

3.2%

4.6%

2.7%

8.3%

8.5%

L-Isoleucine

7.0%

8.5%

5.2%

10.6%

11.1%

L-Methionine

5.1%

6.6%

3.4%

8.3%

10.6%

L-Ornithine

4.9%

6.8%

6.2%

13.2%

12.3%
12.4%

L-Proline

6.6%

8.9%

5.3%

11.6%

L-Serine

3.6%

5.3%

5.2%

8.5%

9.4%

L-Threonine

4.7%

5.0%

4.2%

10.5%

10.5%

Malic acid

5.0%

4.9%

4.4%

6.8%

7.5%

Average

4.8%

6.3%

4.5%

9.6%

10.3%

Conclusion

References

Modifications to the Bravo Metabolomics
Sample Prep Platform that reduced
the required starting plasma volume
from 100 to 25 µL were describedin this application note. Excellent
metabolite recovery with the method
was demonstrated across representative
chemical classes of compounds.
Additionally, the automated method
offers improved reproducibility whencompared to a laboratory environment
where multiple users manuallyprocessed samples.

1.

Automated Metabolite Extraction
for Plasma using the Agilent Bravo
Platform. Agilent Technologies
technical overview, publication
number 5994-0685, 2019.

2.

Discovery Metabolomics
LC/MS Methods Optimized for Polar
Metabolites. Agilent Technologies
application note, publication number5994-1492, 2019.

11www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.
RA44175.4137847222
This information is subject to change without notice.

© Agilent Technologies, Inc. 2020Printed in the USA, December 8, 2020
5994-2156EN