275 / 658
Demonstrating High-Performance Quantitative
Analysis of Benzodiazepines using Multiplexed
SIM with High-Resolution, Accurate Mass
Detection on the Q Exactive LC/MS
Kevin J. McHale; Thermo Fisher Scientific, Somerset, NJ
Introduction
In today’s modern forensic toxicology laboratories,
there is a growing demand to have a mass spectrometer
with the power and flexibility to perform experiments
both for the identification of unknown compounds
and for trace-level quantification of target analytes.
Additionally, this platform must execute these analyses
with minimal sample preparation, provide consistent
results and be easily assimilated into the laboratory
workflows. With the introduction of the Thermo
Scientific Q Exactive high-performance benchtop
quadrupole-Orbitrap mass spectrometer, the most
stringent qualitative and quantitative objectives can be
met. By using high-resolution, accurate mass (HRAM)
detection with quadrupole selected ion monitoring
(SIM), targeted quantification of benzodiazepines in
urine can be accomplished with sensitivity that rivals
triple stage quadrupole instruments in selected reaction
monitoring (SRM) mode.
Goal
To demonstrate the feasibility of high sensitivity
liquid chromatography-mass spectrometry (LC/MS)
quantification of benzodiazepines in urine by combining
multiplexed SIM with high-resolution, accurate mass
detection on the Q Exactive™ high-performance
benchtop quadrupole-Orbitrap mass spectrometer.
Experimental
Sample Preparation
Eight benzodiazepines were spiked into blank human
urine containing acetonitrile at 10% (v/v) from 0.0125
to 250 ng/mL prior to LC/MS.
UHPLC
Ultra high performance LC (UHPLC) analyses were
performed using a Thermo Scientific Accela 1250
liquid chromatography system with an Open Accela™
autosampler. Gradient elution with a Thermo Scientific
Application
Note: 551
Key Words
• Q Exactive
• Accela UHPLC
• Selected Ion
Monitoring
• Drug Quantitation
Hypersil GOLD PFP column (50 x 2.1 mm; 1.9 μm
particle size) was used at a flow rate of 500 μL/min.
The injection volume was 5 μL.
Mass Spectrometry
MS measurements were accomplished on a Q Exactive
mass spectrometer with a heated electrospray ionization
(HESI) source in positive ion mode. Quadrupole
isolation was set to 1.5
m/z
with subsequent detection
at a mass resolution of 140,000 FWHM via external
mass calibration.
Results and Discussion
SIM is a well-established technique for targeted LC/MS
quantitation using single quadrupole mass spectrometers.
However, its utility is limited owing to the low specificity
of unit mass resolution on single quads. The Q Exactive
mass spectrometer, which employs Orbitrap-based high-
resolution, accurate mass detection, overcomes this
limitation. Additionally, the duty cycle on the
Q Exactive MS is enhanced by measuring multiple
SIM ions simultaneously in the Orbitrap mass analyzer.
The process of multiplexed SIM is illustrated in Figure 1.
Four different ions are selected by the quadrupole
and stored in the C-trap while the Orbitrap analyzer
measures the ions from the previous cycle. This process
is repeated by passing the four SIM ions from the C-trap
to the Orbitrap analyzer for the next mass measurement.
The Q Exactive mass spectrometer has the capability to
multiplex between two and ten SIM ions.
Table 1 lists the eight benzodiazepines quantified
by HRAM LC/MS with their multiplexed SIM time
windows, the measured mass errors using external mass
calibration, and the lower limits of quantitation (LLOQs)
in urine on the Q Exactive mass spectrometer. Two key
points to highlight in Table 1 are that (1) mass errors on
the Q Exactive system are significantly less than 5 ppm
without the need of an internal calibration mass, and
(2) the LLOQs of the eight benzodiazepines analyzed in
urine are in the pg/mL range.
Figure 1. Schematic of multiplexed SIM on the Q Exactive mass spectrometer
Figure 1
SIM 1
SIM 2
SIM 3
SIM 4
SIM 1
SIM 2
SIM 3
SIM 4