SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A15
ANALYSIS FOR LORAZEPAM IN BLOOD AND URINE BY GAS CHROMATOGRAPHY ­
MASS SPECTROMETRY - POSITIVE CHEMICAL IONIZATION
David M. Andrenyak·. Center for Human Toxicology, University of Utah, Salt Lake City, Utah 84112,
USA.
Lorazepam is a benzodiazepine drug that is used to treat anxiety and seizures. It is also prescribed as a pre
operative sedative. Because lorazepam is a powerful CNS depressant and can enhance the effect of other
CNS depressants, effective procedures for the analysis of lorazepam in biological samples are essential.
This study reports on the analysis for \orazepam by gas chromatography - mass spectrometry (GC-MS) in
biological samples. Liquid - liquid extraction was performed on 1 mL sample volumes using clean,
separate 16 x 100 mm silanized culture tubes with PTFE lined screw caps. Prior to extraction, the urine
samples were hydrolyzed by adding 1 mL of 5000 units I mL ~-glucuronidase in 0.1 M sodium Acetate, pH
5.0. The urine samples were incubated 3 hours at 40' C in an incubation oven. Following incubation,
internal standard (100 ilL of 1 ng/!lL lorazepam-d 4 ) was added to the urine and unhydrolyzed blood
samples. A 1 mL volume of saturated sodium borate buffer (pH 9) and 5 mL oftoluene: dichloromethane
(70:30) was added to each urine and blood sample. After mixing 20 minutes on a reciprocal shaker, the
tubes were centrifuged at 2000 rpm for 10 minutes. The organic layer was collected into clean, separate 13
x 100 mm culture tubes. The extracts were evaporated to dryness under a stream at air at 40' C using the
Turboevap evaporator. The extracts were derivatized by adding 100 ilL of N,O-bis(trimethylsilyl)
trifluoroacetamide +1% trimethylchlorosilane) to each tube. The extract tubes were heated at 70' C for 20
minutes by using a dry block heater. After heating, the extracts were cooled to room temperature and
transferred to clean, separate autosampler vials. The derivative reaction incorporated 2 trimethylsilyl
(TMS) groups on the lorazepam molecule. The derivatized extracts were analyzed by GC-MS using an
Agilent 5973 GC-MSD system. Chromatographic separation was achieved using an HP-1 MS 30 M x 0.25
mm, 0.33 11m film capillary column (AgiJent) and UHP helium (1 mL/min) as the carrier gas. The column
oven temperature program was 125' C, hold O. 2 minutes, 20· C/ minute to 300', hold 7.5 minutes.
Splitless injection with injection port temperature at 250· C was used. The transfer line temperature was
set at 300' C. Positive chemical ionization was utilized with ammonia as the reagent gas and an ion source
temperature at 200' C. Selected ion monitoring was employed to analyze (mlz) 465 for 10razepam-diTMS
and (m/z) 471 for lorazepam-d 4 -diTMS. The 10razepam-diTMS mass spectrum had a base peak at (m/z)
465 and the 10razepam-~-diTMS mass spectrum had a base peak at (mlz) 469. Since 10razepam-diTMS
had a significant (m/z) 469 ion fragment, the (m/z) 471 ion was used to monitor for the lorazepam-~diTMS.
Calibration standards were prepared with blank bovine blood and ranged from 2.5 nglmL to 1000
ngimL. In house controls were prepared with blank blood at 3.5 nglmL, 100 nglmL, 650 nglmL and with
blank human urine at 100 ngimL. In the intra-assay accuracy evaluation, the lorazepam concentration was
within 13% of target. In the intra-assay precision evaluation, the coefficients of variation were within 8%.
This method was used to analyze blood and urine samples from a post mortem case.
Keywords: Lorazepam , GC-MS, Positive Chemical Ionization
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