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Software Driven Quantitative LC-MS Analysis
of Opioids in Urine for Forensic Laboratories
Kristine Van Natta, Xiang He, Marta Kozak; Thermo Fisher Scientific, San Jose, CA
Application
Note: 541
Key Words
• TraceFinder
Software
• TSQ Quantum
Ultra
• Drugs of Abuse
Introduction
Thermo Scientific TraceFinder software provides an inte-
grated workflow approach for routine forensic screening
and quantitation from method development and data
acquisition to data processing and on through reporting.
The TraceFinder™ software supports all Thermo Scientific
quantitative liquid chromatography-mass spectrometry
(LC-MS) systems with fully integrated support for Thermo
Scientific Transcend multiplexing systems. The software
also provides integrated levels of security from a lab man-
ager to a routine user.
Goal
To demonstrate the software driven quantitative analysis
of six opioids in urine using the Thermo Scientific TSQ
Quantum Ultra mass spectrometer and TraceFinder
software.
Experimental
Sample Preparation
Urine was spiked with internal standards and hydrolyzed
with
β
-glucuronidase. Fisher Chemical Optima
®
LC/MS
Methanol was added to the hydrolysis mixture and the
resulting mixture was centrifuged. The supernatant was
further diluted and subjected to LC-MS analysis.
LC-MS/MS conditions
LC-MS analysis was performed on a TSQ Quantum
Ultra™ mass spectrometer equipped with a heated
electrospray ionization (HESI) probe coupled with a
Transcend™ TLX system operating in LX mode. Two
selected reaction monitoring (SRM) transitions were
monitored for each compound. High pressure liquid
chromatography (HPLC) was carried out on a Thermo
Scientific Hypersil GOLD aQ column (50 × 4.6 mm, 1.9
µm particle size) at 30 °C. The MS source conditions were
as follows:
Spray Voltage
3500 V
Vaporizer Temp
350 °C
Sheath Gas
80 (arbitrary units)
Ion Sweep Gas
0 (arbitrary units)
Aux Gas
5 (arbitrary units)
Capillary Temp
250 °C
Software
TraceFinder software was used for method development
and routine analysis during validation.
Main Tabs in TraceFinder
Figure 1 shows the four main tabs in TraceFinder software:
Acquisition, Data Review, Method Development and
Configuration.
Compound Data Store
Figure 2 shows the Compound Data Store (CDS) for this
opioid application. Entries of the analytes in this CDS
contain the quantifier ion, qualifier ion and retention times
for easy addition to a Master Method.
Master Method
The Master Method contains all of the information needed
for an assay including that for instrument acquisition,
data processing and reporting. The five main categories of
information are: General (including assay type, injection
volume, and instrument method), Compound (including
acquisition list selected from the CDS, detection param-
eters, calibration and control levels), Flags, Groups and
Reports. Selected tabs in the General and Compounds
sections are shown in Figure 3. Many flagging parameters
are available to customize data review and reports. Some
of these parameters are shown in Figure 4.
Instrument Method
Instrument methods including autosampler, HPLC, and
mass spectrometer parameters can be directly edited within
TraceFinder 1.1 through a Thermo Scientific Xcalibur
software interface.
Batch
Creating a batch involves assigning a project, linking to
the master method, building a run-sequence and finally
submitting the batch. Multiplexing channels are also con-
trolled in the batch creation as seen in Figure 5.