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Quantitative LC-MS Analysis of 14
Benzodiazepines in Urine Using TraceFinder 1.1
Software and High Resolution Accurate Mass
Xiang He, Marta Kozak; Thermo Fisher Scientific, San Jose, CA
Introduction
Thermo Scientific TraceFinder 1.1 software is developed
for quantitative analysis for clinical research laboratories.
The software is designed for routine data acquisition,
quantitation, qualitative screening and reporting on
all Thermo Scientific liquid chromatography mass
spectrometry (LC-MS) systems, including high resolution
accurate mass (HRAM) instruments, with fully integrated
support for the Thermo Scientific Transcend multiplexing
system.
TraceFinder
TM
1.1 quantitative software simplifies
routine analysis for the operator by executing a stepwise
workflow from batch creation to reporting. For clinical
research laboratories employing multiple types of LC-MS
systems, TraceFinder 1.1 software eliminates the need to
learn and maintain multiple software programs.
TraceFinder 1.1 software provides many easy
approaches to execute workflow routines for operators
and lab managers. The work presented here demonstrates
the workflow used by lab managers during method
development and includes processing method creation
using the compound data store (CDS). The operator’s
workflow includes batch submission, real time monitoring,
data review and report generation.
Goal
To demonstrate a new, easy-to-use workflow-driven
quantitative method for 14 benzodiazepines in urine
using the Thermo Scientific Exactive high performance
benchtop mass spectrometer and TraceFinder 1.1 routine
quantitative software.
Methods
Sample Preparation
Urine was spiked with internal standards and hydrolyzed
with beta-glucuronidase. Acetonitrile was added to the
hydrolyzed sample and the resulting mixture was centri-
fuged. Supernatant was further diluted and subjected
to LC-MS analysis.
LC-MS/MS conditions
LC-MS analysis was performed on an Exactive
TM
mass
spectrometer with a heated electrospray ionization (HESI)
source coupled with a Transcend™ TLX system used in
LX mode. Full scan mass spectrometry analysis was done
with resolution of 100,000 (FWHM at
m/z
200) with a
mass isolation window of 3 ppm. Exact mass was used
for compound identification. High performance liquid
chromatography (HPLC) was carried out on a Thermo
Scientific Hypersil GOLD PFP column (100 × 2.1 mm,
5 µm particle size) at room temperature.
The MS conditions were as follows:
Ionization
HESI-II
Polarity
Positive
Vaporizer temp (°C)
350
Capillary temp (°C)
350
Spray voltage (V)
3500
Sheath gas (AU)
40
Auxillary gas (AU)
10
Data acquisition mode
Full scan
AGC target
1.00E+06
Lock mass
(m/z)
279.2591
Scan range
(m/z)
135-600
Max injection time (ms)
100
Resolution
100,000
Software
Method development, data acquisition, data processing
and report generation were all executed in TraceFinder 1.1
routine quantitation software.
Results and Discussion
Streamlined Workflow:
The entire workflow in TraceFinder 1.1 software is easy to
set up and is summarized in Figure 1.
Application
Note: 529
Key Words
Software
CDS
Master Method
Instrument Method
Batch
Data Acquisition
Real Time Status
Data Review
Report
Figure 1
Figure 1. TraceFinder 1.1 workflow for quantitative analysis