True High Throughput Bioanalysis Using the Echo® MSSystem
Rapid Sample Analysis Using AEMS Technology Improves Bioanalytical Study TurnaroundRolf Kern1, Chang Liu2
1
SCIEX, USA, 2SCIEX, CanadaAnalysis of biological samples to assess the pharmacokinetic(PK) properties of drug candidates is a cornerstone assay that isroutinely performed in virtually all pharmaceutical companies.
Despite the importance of PK information in drug discovery anddevelopment, bioanalytical data is time consuming data toproduce. Many individual time points must be analyzed, often
from multiple subjects, to generate data for a single compound.
When considering the time required to prepare samples, evenjust using a simple protein precipitation, in addition to 1 to 2
minute individual sample analysis times in the quickest cases,
data is generally not available until the day after the bioanalyst
begins work on a study.
The SCIEX Echo® MS System, which uses Acoustic Droplet
Ejection technology and an Open Port Interface (OPI) coupled tothe high-sensitivity SCIEX Triple Quad™ 6500+ Mass
Spectrometer, is capable of dramatically reducing analysis time,
as well as reducing the need for sample preparation.1 This
allows PK study data to be available the day samples areprepared, giving researchers faster access to this important data(Figure 1).
Selection of the most appropriate sample preparation method isalways a balance of required sensitivity, necessary throughput
and the cost of consumables like SPE cartridges. For
bioanalytical assays where sensitivity requirements are easily
met, faster analysis and simpler sample prep lead to a more
efficient overall process. Here, very simple sample preparation
techniques were explored in combination with the SCIEX Echo®
MS System for rapid generation of routine bioanalysis studies.

Key features of Echo MS System for routinebioanalysis• Integral to the Echo MS System is the high-sensitivity SCIEXTriple Quad 6500+ System for meeting sensitivity
requirements• Good quantification limits were achieved with all three simple
sample preparation techniques that were explored, meetingthe needs of many routine bioanalysis studies for throughput
and cost• Very high reproducibility of analysis obtained even out of
untreated plasma

Figure 1. Complete pharmacokinetic study in plasma in under 5minutes. A calibration curve of fentanyl in untreated plasma wasgenerated across 16 different concentrations with 5x replicates at
each point in under 5 minutes.

• Exceptional speed and low carryover of Echo MS System
combined with simplified sample preparation demonstrated
here make this an efficient and fit-for-purpose bioanalysis
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Methods
Sample preparation: Fentanyl (Cerilliant) working standards
were prepared by serial dilution in 10% v/v methanol in waterfrom 65.536 µg/mL to 0.002 µg/mL in 1:1 increments.
Norfentanyl (Cerilliant) was used as an internal standard, andwas diluted to 200 ng/mL in 10% methanol in water.
10 µL of working standard and 10 µL of internal standard wasadded to 200 µL of K2EDTA Sprague-Dawley rat plasma(Bioreclamation IVT). Identical calibrations were also prepared in
0.1% and 0.01% by volume PEG400 (Sigma) in rat plasma.
Protein precipitation: 100 µL of prepared plasma was transferred
to a conical bottom 96 well plate. 100 µL of methanol was addedto each standard, the plate was covered and mixed gently on anorbital plate shaker for 5 mins. The plate was centrifuged at 4000rpm for 10 mins to settle the precipitate. 60 µL of supernatant
was transferred to an Echo 384 well plate (Beckman Life
Sciences 384PP 2.0 Microplate) for analysis.
1:1 dilution: 30 µL of the prepared plasma samples wastransferred to an Echo 384-well plate. 30 µL of water was added
to the samples, and the plate was gently mixed on an orbital
shaker for 5 mins.
Untreated plasma: 60 µL of the prepared plasma samples weretransferred to Echo Qualified 384-Well Polypropylene Microplate
2.0 (384PP 2.0).
Prior to analysis, the final sample plates were centrifuged at
3000 rpm to remove any bubbles in the wells, and then briefly
mixed on an orbital shaker to ensure formation of a stable
meniscus in each sample well.
Mass spectrometry: MRM data was collected in positive
polarity using the SCIEX Triple Quad 6500+ System using theparameters outlined in Table 1. Key ion source parameters are
outlined in Table 2. Note that the value for GS1 value is 90 for
this assay, this value is required to be higher than usual because
the GS 1, or nebulizing gas flow, is what provides the aspirating
force that pulls the carrier solvent from the OPI into the ionsource.

Table 1. MRM parameters.
Compound Q1

Q3

Fentanyl

337.2

Norfentanyl

233.0

DwellDPEP

CE

CXP

188.1 4570

1030

1184.1

55

1024

545

Table 2. Ion source settings.
Parameter

Setting

GS 190

GS 270

CUR

30

CAD

12Temp350

IS

5000

Acoustic droplet ejection methods: Acoustic droplet ejection
methods, which are analogous to HPLC methods for the Echo
MS System, contain relatively few parameters.
Flowrate will generally be based on the viscosity of the carrier
solvent , which was pure methanol for this study.
Fluid class will be based on the viscosity of the sample liquid; SPfor liquids less viscous than water, AQ for samples in liquids withviscosity equal to or greater than water.
Delay time can be added to an acquisition to allow for ejectionsof “richer” matrices to come completely back to baseline betweensampling. For bioanalytical work, an additional 1000 msec delay
time was used to ensure high concentration samples did notelevate the baseline of subsequent low concentration samples.
More aqueous or solvent based samples do not generally require
this.
Droplet count is the number of droplets pulsed in very shortsuccession as a single ejection and is analogous to an injection
volume in HPLC work. Optimal droplet count will depend on thematrix and required sensitivity. Richer matrices can show
diminishing returns at higher droplet counts as suppressive
effects and peak broadening counteract the greater amount ofanalyte introduced into the liquid stream. Cleaner matrices allow
for often significant increases in sensitivity (up to 20 droplets insome cases). When assays are being developed, running adroplet ladder is helpful to select the optimal droplet count.
Figure 2 shows a droplet ladder experiment run using a standard
from the protein precipitation calibration. In Figure 2, a linear
signal increase is observed for 1 through 5 droplets, with theresponse leveling off between 8 and 9 droplets. 8 droplets waschosen for this matrix type.

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Figure 3. Example calibration curve for fentanyl in untreatedplasma. Concentration curve from X to Y ng/mL of fentanyl in untreatedplasma was generated and run with n=5 ejections each concentrationlevel.
123456789Figure 2. Droplet ladder for the samples prepared by protein
precipitation. Performing a droplet ladder experiment during assay
development allows the user to balance signal with matrix effects. Herethe x axis is droplet count (n=3 replicates per count) for fentanyl inprotein precipitated plasma. Eight droplets was the optimal number
determined here.

Table 3 below lists the parameters for each acoustic droplet
ejection method for the three different sample preparationexperiments.
Table 3. Acoustic droplet ejection method parameters.
Parameter

Plasma1:1 Dilution

PPT

Flowrate

350

350

350

Fluid ClassAQ

AQ

SP

Additional Delay

1000 ms

1000 ms

1000 msDroplet Count

548Sensitivity achieved with different sample
preparation methods
Three different sample preparation methods were explored, tocharacterize the impact of matrix complexity on the quality ofdata generated. The concentration curves for fentanyl weregenerated in each and the lower limits of quantification (LLOQ),
linearity and reproducibility were determined. Figure 3 shows anexample curve for fentanyl in untreated plasma. Blanks wereperformed before the lowest concentrations ejections to confirm
the LLOQs (Figure 4). Overall, the best performance wasobserved for the data obtained from the untreated plasma.

Data processing: Once acquisition of a batch is complete, a filesplitting algorithm automatically runs which creates individual
files within the acquired .wiff2 file that can be processed in
SCIEX OS Software Analytics module. In high-throughput
environments, where calculations are done by a LIMS or othersoftware, peak areas can automatically be generated if aProcessing Method is specified in the batch. For this example,
the data is processed the same as standard bioanalytical data inAnalytics.
Figure 4. Signal for fentanyl at lower limit of quantification. Blank
samples followed by standard ejections of the fentanyl in untreatedplasma at a concentration of 0.2 ng/mL, demonstrating good signal at
the LLOQ.
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Table 4. Reproducibility of the calibration curves in eachsample preparation strategy. The %CV for n=5 reps for eachcalibration level was calculated, with the majority being below 5%
across the concentrations.
Protein
1:1 Plasma: Water
Precipitation

Table 5. Matrix effects observed for different PEG400
concentrations. Standards (top row) and QC Samples prepared at the102.4 ng/ml level. Values are of n=5.

MatrixPeak Area %cv

AverageCalculatedConcentration

AverageAccuracy

Std Conc.

Plasma

0.1

BLOQ

BLOQ

BLOQ

Plasma, no PEG400

7.7

104.8

102.4%

0.2

9.4

BLOQ

BLOG

Plasma w/ 0.01%
PEG400 by volume

5.3

99.4

97.1%

0.4

7.6

10.6

9.5

0.8

5.5*6.4

Plasma w/ 0.10%
PEG400 by volume

3.2

76.7

74.9%

1.6

10.3

5.6

7.6

3.2

3.5

8.1

6.9

6.4

3.9

3.9

4.7

12.8

3.8

8.8

1.9

25.6

6.8

4.2

4.8

51.2

2.4

3.9

6.6

102.4

7.7

3.4

2.9

204.8

1.8

7.2

6.1

409.6

4.2

3.9

4.9

819.2

3.9

3.7

6.9

1638.4

2.8

4.4

5.2

3276.8

3.2

3.2

5.9

* Standard was lost during transfer
BLOQ Standard was below the limit of quantitation

Average accuracy for all replicates across the concentration
curves was between 85 and 115% of nominal for all preparation
methods. The reproducibility at all concentration levels for eachsample preparation technique is listed in Table 4. It is worth
noting that for all three preparations, there was not a significant
difference in either assay sensitivity or variability.

Evaluation of suppression effects
In addition to sensitivity, reproducibility and linearity, the effect ofpotential matrix suppression was also investigated by analyzing
standards prepared in plasma fortified with PEG400, which weretreated as quality controls when the data was processed.
PEG400 is a common formulation agent used in pharmaceuticalresearch that can have significant suppressive effects inelectrospray mass spectrometry. Stable labeled internal
standards generally compensate for ionization suppression, as
long as the analyte is present at a level high enough above thelower limit of quantification to still be measurable. In research PK
work, labeled internal standards are not always available, so

chemically similar compounds are often selected for use. Even
when chemically similar, ionization suppression does not always
effect analytes and internal standards equally, especially if theyare chromatographically resolved. In this case norfentanyl, which
is a metabolite of fentanyl, was used as an IS and there is nochromatography to consider. The effects of different levels ofPEG400 in untreated plasma can be seen in Table 5 at the 102.4
ng/mL level as an example. The deviation from nominal at otherconcentrations was very similar to the 102.4 level for the two
PEG400 levels.

Conclusions
The goal of this study was to assess the feasibility of the Echo
MS System for routine, early stage bioanalytical quantification.
Three plasma sample preparation techniques were explored toevaluate the effect minimal sample preparation would have on
the quality of data produced. The approaches used in this projectshow minimal difference for dilution, basic protein precipitationand simply analyzing plasma directly without any treatment at all.
For routine analysis, where the assay sensitivity requirements
demonstrated here are sufficient, the speed and simplicity ofanalysis using the Echo MS System offers an attractive
approach for high turnaround bioanalytical laboratories.
In future work, the use of more thorough preparation techniquessuch as SPE, will be explored for the impact on sensitivity. Inaddition, the use of labeled internal standards will also beexplored to further improve the data quality.

p4References
1.

Rapid MS/MS analysis with Acoustic Ejection Mass
Spectrometry (AEMS) - Using the SCIEX Echo® MS System
to break bottlenecks in quantitative mass spectrometry
throughput. SCIEX technical note RUO-MKT-02-11385-A.

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