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Quantitation of 14 Benzodiazepines and
Benzodiazepine Metabolites in Urine Using
a Triple Stage Quadrupole LC-MS System
Kristine Van Natta, Marta Kozak; Thermo Fisher Scientific, San Jose, CA
Introduction
Benzodiazepines have a broad range of therapeutic
use and are widely prescribed as safe drugs for the
treatment of insomnia, anxiety and seizures and for their
amnesic effects prior to medical procedures. They are
also abused for their psychoactive effects, in suicide and
in drug-facilitated sexual assault. Simple, robust and
precise analytical methods are needed to quantitate these
compounds in biological matrices for forensic purposes.
Goal
To develop a specific and robust dilute and
shoot quantitative method for the analysis of
14 benzodiazepines and metabolites in urine.
These compounds include: 2-hydroxyethylflurazepam,
7-aminoclonazepam, 7-aminoflunitrazepam,
7-aminonitrazepam,
α
-hydroxyalprazolam,
α
-hydroxytriazolam, alprazolam, desalkylflurazepam,
diazepam, lorazepam, midazolam, nordiazepam,
oxazepam and temazepam.
Methods
Sample Preparation
Urine was spiked with internal standards and
hydrolyzed with
β
-glucuronidase. Deuterated analog
internal standards were used for all compounds except
α
-hydroxytriazolam and lorazepam. Isotopic contribution
from the di-chlorinated parent interfered with the d4
internal standards. Deuterated
α
-hydroxyalprazolam
and oxazepam, respectively, were used instead. After
hydrolysis, methanol was added to the hydrolysis mixture
and the resulting mixture was centrifuged. Supernatant
was further diluted and subject to LC-MS analysis.
Application
Note: 547
Key Words
• TSQ Quantum
Ultra
• Forensic
Toxicology
HPLC Conditions
Chromatographic analysis was performed using Thermo
Scientific Accela 600 HPLC pumps and a Thermo
Scientific Hypersil GOLD aQ column (50 x 4.6 mm,
1.9 µm particle size). The total run time was 6.5 minutes.
MS Conditions
MS analysis was carried out on a Thermo Scientific TSQ
Quantum Ultra triple stage quadrupole mass spectrometer
equipped with a heated electrospray ionization (HESI-II)
probe. Two selected reaction monitoring (SRM) transitions
were monitored for each compound to provide ion ratio
confirmations (IRC).
The timed selected reaction monitoring (T-SRM)
was used. T-SRM allows the instrument to scan only
for those compounds that are expected to be eluting
at a certain time. The data for a particular target
compound is acquired only in a short window around
the known retention time, not throughout the entire run.
Using T-SRM significantly reduces the number of SRM
transitions that are monitored in parallel at a certain
retention time. At a constant acquisition rate (cycle time)
a significantly longer scan time (dwell time) is available
for each transition resulting in higher sensitivity and
lower quantitation limits, improved RSDs and more data
points per chromatographic peak.
Validation
Standard curves were prepared by fortifying pooled blank
human urine with analytes. Quality control (QC) samples
were prepared in a similar manner at concentrations
corresponding to the low, middle and high end of the
calibration range. Intra-run variability and robustness
were determined by analyzing six replicates of each
QC level with a calibration curve. Matrix effects were
investigated by preparing samples in 8 different lots of
human urine at twice the limit of quantitation (LOQ) of
the method and monitoring peak area recovery compared
to samples prepared in water.