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Figure 1
MRM transitions of 27 benzodiazepines, 3 nonbenzodi-
azepine hypnotics, and two internal standards on the Allure® PFP
Propyl column.
•
20
•
2008 vol. 1
Clinical/Forensics
Benzodiazepines are widely prescribed drugs used
for treating anxiety and sleep disorders. Since
addiction and abuse can occur, efficient screening
methods are critical to clinical, forensic, and toxi-
cology laboratories. The liquid chromatography
tandem mass spectrometry (LC/MS/MS) method
presented here offers several advantages over other
techniques: minimal sample preparation, fast
analysis times, multiple reaction monitoring tran-
sitions for quantification and confirmation, and
sensitivity down to 0.10-10ng/mL. This method
uses the Allure® PFP Propyl stationary phase,
which retains compounds long enough to minimize
matrix interferences and chromatographically sepa-
rate compounds that share the same precursor ion.
Procedure
Samples were prepared by adding 100µL of inter-
nal standard solution (1µg/mL D5-Diazepam and
D3-Dioxepine) to 100µL urine, diluting with
800µL LC grade water, and centrifuging. The sam-
ples were then analyzed by LC/MS/MS.
Compound separation was achieved using an
Allure® PFP Propyl column and a mobile phase
gradient program.
A 3200 QTrap® LC/MS/MS system equipped with
a Turbo V™ source with electrospray ionization
was used to develop and detect the two MRM
transitions (Table 1). For each compound, MRM
1 was used to quantify, and the ratio to MRM 2
was used to confirm.
Cliquid™Drug Screen & Quant Software was used
to process data and generate automatic reporting
relevant to forensic guidelines. Limits of quantifi-
cation were determined and the automated
reporting allowed for positive confirmation based
on the detected MRM ratios.
Results
By diluting the urine samples ten-fold, matrix
effects are reduced (reducing ion suppression) and
LOQs between 0.10ng/mL and 10ng/mL can be
achieved (Table 1). Ion suppression is further
reduced by using a retentive column which 1)
elutes matrix interferences before the compounds
of interest, and 2) allows for better desolvation
efficiency due to the ability to use 90% organic in
the mobile phase composition. The Allure® PFP
Propyl is such a column; it has high retention and
selectivity for basic drug compounds, such as ben-
zodiazepines (Figure 1).
Fast, Sensitive Analysis of Benzodiazepines by LC/MS/MS
Quantify an Order of Magnitude below Typical Methods
By Kristi Sellers, Clinical/Forensic Innovations Chemist
• Achieve full chromatographic separation of compounds with shared precursor ions
• Quantify compounds at 10ng/mL or less in urine.
• Increase accuracy with improved desolution efficiency from highly organic mobile phase.
Sample:
benzodiazepines
Inj.: 20µL
Conc.: NA
Solvent: NA
Column:
Allure
®
PFP Propyl
Cat.#: 9169552
Dimensions: 50mm x 2.1mm
Particle size: 5µm
Pore size: 60Å
Conditions:
Mobile phase: A: 0.1% formic acid and 1mM
ammonium formate in water
B: 0.1% formic acid and 1mM
ammonium formate in acetonitrile
Time
Flow %B
(min.)
(µL/min.)
0.0
500
10
10.00
1000
90
15.00
1000
90
15.50
500
10
17.50
500
10
Flow:
see gradient table
Temp.:
40°C
Det.:
Applied Biosystems/MDS Sciex
API 3200™ MS/MS system
Ion Source: Electrospray, positive
IonSpray Voltage: NA
Gas 1: NA
Gas 2: NA
Source Temperature: 500°C
Data courtesy of: Applied Biosystems MDS Sciex
LC_PH0463
Analyze 27
benzodiazepines
in less than
10 minutes!