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Quantitation of 12 Benzodiazepines and
Metabolites in Urine Using Ultrahigh Resolution
LC-MS for Forensic Toxicology Use
Kent Johnson, Fortes Laboratories, Portland, OR; Marta Kozak, Thermo Fisher Scientific, San Jose, CA
Goal
To demonstrate the quantitation of 12 benzodiazepines in
urine using a liquid chromatography-mass spectrometry
(LC-MS) method and ultrahigh resolution with the
Thermo Scientific Exactive benchtop mass spectrometer
for forensic analysis.
Experimental
Standards and Samples Preparation
Calibration standards were prepared by spiking blank
urine with 12 benzodiazepines (lorazepam, nordiazepam,
oxazepam, temazepam, hydroxytriazolam, 7-
aminoclonazepam, 7-aminonitrazepam,
hydroxyalprazolam, 7-aminoflunitrazepam,
desalkylflurazepam, diazepam, and 2-
hydroxyethylflurazepam) to final concentrations ranging
from 10 ng/mL to 2,000 ng/mL.
Calibration standards and urine samples were spiked
with internal standards (10 deuterated benzodiazepines),
hydrolyzed and processed using a solid phase extraction
(SPE) procedure.
Third party QC samples containing 6 benzodiazepines
were processed and analyzed to obtain method accuracy
and precision.
HPLC
HPLC analysis was performed using a Thermo Scientific
Accela liquid chromatography system with a Thermo
Scientific Hypersil GOLD PFP column (50 x 2.1 mm;
5 µm). A processed sample of 5 µL was analyzed with a
6-minute gradient method.
Mass Spectrometry
MS analysis was carried out on an Exactive™ benchtop
LC-MS instrument with an electrospray ionization (ESI)
source. Full scan data with resolution of 100,000
(FWHM) was acquired.
Results and Discussion
Figure 1 displays 6 of the 12 selected benzodiazepines at
10 ng/mL and internal standards. Chromatograms for
compound detection and quantitation are reconstructed
with a mass tolerance of 5 ppm.
Figure 2 shows the calibration curve for this set. Data
results for the other six benzodiazepines are available
upon request.
Conclusion
The Exactive benchtop LC-MS instrument provides
excellent quantitative analysis of 12 benzodiazepines, from
10 ng/mL to 5000 ng/mL in urine, using ultrahigh
resolution full scan data acquisition in a 6-minute method.
The accuracy, precision, LOQ, and linearity range of the
method meet the demands of today’s forensic toxicology
laboratories.
Method Performance Summary
senipezaidozneB
setylanA tegraT
enirU
xirtaM
Limit of Quantitation (LOQ)
10 ng/mL
%58 >
yrevoceR
Lm/gn 0005 – Lm/gn 01
ytiraeniL yassA
%4 <
)VC%( noisicerP
Carryover at Lower Limit of Quantitation (LLOQ) < 1%
Lm 2
emuloV elpmaS
setunim 6
emiT sisylanA
Analyte
Mean Conc.(ng/mL)
% Recovery %RSD
Oxazepam
248
99.3
1
Nordiazepam
234
93.5
1.4
Temazepam
218
87.1
4
Desalkylflurazepam
214
85.7
4
Lorazepam
227
90.8
0.4
Hydroxyalprazolam
255
102
0.4
Key Words
• Exactive
• Accela HPLC
• Pain
Management
• Forensic
Toxicology
Application
Note: 489b
Figure 1: Chromatograms of 6 of the 12 selected benzodiazepines at
10 ng/mL and internal standards.
Figure 2: Calibration curves (10-5000 ng/mL) for all analytes
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