Page 6 - CRFTApplication Compendium_090613

2 Development of a Method for Evaluation of Mass Spectrometer Performance in Real Time
Overview
Purpose: To develop a method which monitors system performance while
simultaneously performing quantitation. The research method is used for verification of
the internal standard (IS) precursor and product ions. The Internal Standard Verification
(ISV) method can be used as any quantitative method would be used. Results of the
method are available in real time to immediately determine the confidence that can be
placed in the data or to initiate follow-on actions.
Methods: First, a series of testosterone injections are used to demonstrate generation
of the confidence data while performing quantitative analysis. Next, conditions are
imposed on the mass spectrometer which give rise to fault conditions (mimicking
resolution or mass drift) to demonstrate the ability to identify data that is suspicious or
incorrect.
Results: The ISV method is shown to have the ability to simultaneously monitor the
calibration state or fault state of the system while maintaining the ability to quantitate
linearly down the LOD.
Introduction
Manual data review has become a common step in the release of results, this can
occupy significant time and effort for a skilled mass spectroscopist. Usually an
abnormally low or high result will trigger the need for review and the recourse is usually
to manually review the quality of the chromatographic peaks for the various analytes.
Here we introduce a method that simultaneously monitors the operation of the mass
spectrometer, including resolution, peak width and transmission, during the analytical
assay without any sacrifice to analytical performance. This method utilizes the internal
standard and does not require any additional steps in sample preparation. The resulting
data can speed up manual review and may reduce or eliminate the need for manual
review.
While performing routine sample analysis, one can monitor one or two ions to evaluate
the performance of the mass spectrometer. However, these monitor ions must be
predictable ions, with a relatively intense response, they should not require additional
sample handling and should avoid causing negative effects on the measurement of any
low concentration anaytes. In most analysis, the internal standard is present as a
relatively strong and predicable ion but it is underutilized. A new mode of operating a
triple stage quadrupole (TSQ) has been developed that uses the internal standard to
evaluate Q1 and Q3 mass position, peak width and intensities, in real time, during
routine analysis to alert the user whenever any of these parameters are out of tolerance.
Methods
Sample Preparation
Samples of testosterone and testosterone d3 were made up in 50/50 MeOH/Water. For
the purpose of this study the Internal standard concentration in all samples was 0.8
pg/uL.
Liquid Chromatography
Considering the samples were made up in neat solutions, a simple 3 min
chromatographic method was used to produce a 3 sec peak, which is representative of
anticipated analytical conditions.
Mass Spectrometry
Samples were run using a Thermo Scientific™ TSQ Endura™ triple-stage quadrupole
mass spectrometer. The method was developed by starting with a standard
Testosterone method that identifies the analyte ions (Testosterone: 289.4 97.2, 109.2)
and their respective internal standard (Testoserone d3: 292.497.2).
To develop the ISV method from a standard method, the IS is first identified for ISV. The
precursor and product ions mass positions are taken from the IS SRM. The resolution
value and tolerances for resolution and mass position are automatically populated. The
user must add the expected intensity for the parent, product and SRM transitions.
Adjustments can be made to all tolerance values as required. The user also has control
over the amount of scan time that is taken from the IS transition to perform the ISV
scans. Figure 1 shows the method parameters in the TSQ Endura MS method editor.
Figure 2 shows the summary of the method, including the ISV parameters.
When the ISV method is gene
specified, the method UI adds
about the precursor ion and b)
the product ion mass. The sca
taken from the internal standa
This method shown here has
are specified across the peak.
transitions will have ~100 ms
unaffected and have ~100 ms
time perform the ISV scans. It
well understood, strong transit
20 to 80% of the IS dwell time
In the present case 80% of th
dwell time and both the precur
product ion mode (292.4 95.
Data Analysis
Data was collected using Xcali
worked up using standard stat
FIGURE 1. TSQ Endura MS
Testosterone analysis using
FIGURE 2. TSQ Endura MS
Results
Quantitation and Monitoring
Routine sample analysis typic
analyte ion and monitoring a s
These analyses can also have
standards. To reduce complexi
method, a simple testosterone
internal standard was used to
spectrometer through a series
monitored across the useful a
used to both normalize the sy
the calibration of the mass sp
Figure 3 shows the calibration
using the same method witho
that including the ISV portion i