SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

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RAPID, SIMPLE AND V ALIDA TED GC-MS ASSAY FOR DETERMINATION OF DRUGS
RELEVANT IN DIACNOSIS OF BRAIN DEATH IN HUMAN BLOOD PLASMA
Frank T. Peters', Julia Jung, Thomas Kraemer and Hans H. Maurer
Department ofExperimental and Clinical Toxicology, University of Saarland, D-66421 Homburg (Saar),
Germany, frank.peters@uniklinik-saarland.de
Background: Before declaring the brain death of a patient (e.g. prior to explantation of organs), a ~umber
of requirements have to be fulfilled. One of them is the exclusion of effective plasma concentratIOns of
drugs which might mimic brain death, especially ofthose typically administered in intensive Cl"e medicine.
Recommendations for toxicological analysis in the context of the diagnosis of brain death have recently
been published (Hall bach et a\., T1AFT Bulletin 34, 2004, 14-16), which include a minimum consensus on
the relevant analytes (thiopental, pentobarbital, methohexital, phenobarbital, diazepam, nordiazepam, and
midazolam). The proposed limits of quantification correspond to one half of their lowest therapeutic
concentrations. Therefore, the aim of the presented study was to develop and validate a rapid and simple
assay for determination ofthe above mentioned drugs in human plasma samples.
Methods: After addition of 50 J.1l of internal standard solution (4.0 mg/l pentobarbital-ds, 2.0 mg/l
methohexital-ds, 40.0 mg/l phenobarbital-ds, 0.8 mg/l diazepam-ds, and 0.8 mg/l nordiazepam-ds in butyl
acetate) and 50 J.11 of butyl acetate to 200 III of plasma, the samples were extracted for 2 min on a rotary
shaker. After phase separation by centrifugation (1 min, 10000 g), 2 III of the organic phase (upper) were
injected into the GC-MS system (Agilent, GC-MSD 5973). The analytes were separated within 10 min by
gas chromatography (HP-l column, 12 m x 0.2 mm LD.) and detected by mass spectrometry. The mass
spectrometer was operated in the full scan mode for identification and in the selected ion mode (S1M) for
quantification (target ions ml= 156, 161, 172,247,252,204,209,256,261,242,247,310). Validation was
performed according to a minimum consensus on method validation in this context currently developed by
the Clinical Toxicology Committee of the GTFCh. This included evaluation of selectivity, calibration
model, precision and accuracy. Furthermore, the results for accuracy and precision obtained with six-point
and one-point calibration were systematically compared. Finally, the applicability of the described assay
was tested by analysis ofreal samples from brain death cases.
Results: The analytes were fully separated and sensitively detected. No interfering peaks were detected in
blank plasma samples from ten different sources. The assay was linear from 0.25 to 10 mg/I for
pentobarbital and thiopental, from 0.125 to 10 mg/l for methohexital, from 2.5 to 50 mg/I for phenobarbital,
from 0.05 to 2.5 mg/I for diazepam and nordiazepam, and from 0.01 to 0.5 for midazolam. Using six-point
calibration, accuracy data (in terms of bias) ranged from -17.9% to 23.7%. Within-day and intermediate
precision data (expressed as CV) ranged from 1.4% to 6.4% and from 2.6% to 7.0%, respectively. Using
one-point calibration with a calibrator close to the center _of the. linearity range, accuracy data (in terms of
bias) ranged from -11.6% to 29.7%. Within-day and intermediate precision data ranged from 1.3% to 6.2%
and from 2.6% to 9.6%, respectively. Recoveries ranged from 85% to 109%. The acceptance criterion
defined by the Clinical Toxicology Committee of the GTFCh (99% confidence interval of measured mean
within ±50% oftarget value) was easily fulfilled for all analytes, even with one-point calibration. The assay
was successfully applied to analysis ofreal brain death cases.
Conclusion: The described assay allows rapid, fast and reliable determination of analytes relevant in the
diagnosis of brain death. Systematic studies showed that the assay can be performed with one-point
calibration. This is an important advantage, because it keeps the workload low for the usually single cases
and because results are needed quickly in this context.
Keywords: brain death, GC-MS, validation
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