SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A49
AN LC-MSIMS METHOD FOR THE QUANTIFICATION OF BUPRENORPHINE AND
NORBUPRENORPHINE IN BLOOD AND URINE
Merja Gergov*, Pirjo Tiainen and I1kka Ojanperli
Department of Forensic Medicine, University ofHelsinki, P.O. Box, 40, FIN-00014 University of Helsinki,
Finland
Background: The synthetic opioid buprenorphine has become common in the maintenance therapy of
opioid addicts in Finland since 1999. The increasing number of samples has required a simple and reliable
analytical method suitable for routine use. LC-MS/MS provides high selectivity and sufficient sensitivity
for the quantitation ofbuprenorphine and its metabolite norbuprenorphine in urine and blood.
Methods: Urine samples were hydrolyzed with ~-glucuronidase enzyme. Sample work-up for blood and
hydrolyzed urine involved liquid-liquid extraction with ethyl acetate at pH 7. The extracts were run by LC
on a Genesis CIS reversed phase column using acetonitrile-ammonium acetate mobile phase at pH 3.2. The
mass spectrometric analysis was performed with a triple quadrupole mass spectrometer equipped with a
turbo ion spray interface in positive mode using multiple reaction monitoring (MRM). Sufficient
sensitivity was achieved only by monitoring the surviving parent ions both for buprenorphine (mlz 468.0)
and norbuprenorphine (mlz 414.2) at the collision energy of 20 eV. Quantification was performed using
deuterated internal standards and four-point calibration.
Results: Validation curves were prepared with four replicates at ten concentration levels. The linearity
criterion was accuracy better than ± 20% at the calibration curve. The method exhibited good linearity
from 1 to 200 /lg/l for urine (r2 ~ 0.996, quadratic regression) and from 0.2 to 100 !lg/l for blood (r ~
0.998, linear regression through zero). The average intra-day precision of real autopsy samples was 3 %
for both compounds in urine, and 7% for buprenorphine and II% for norbuprenorphine in blood. Criteria
for the LOQ were: 1) precision better than ± 20%, 2) inaccuracy lower than ± 25% in urine and 20% in
blood, and 3) signal-to-noise ~ 10. The LOQs for both compounds were I /lgll in urine and 0.2 /lg/l in
blood. In the study of uncertainty of measurement, systematic errors were not observed. The expanded
uncertainty of measurement (at the confidence level of 95%) contained only random errors being for both
compounds 15% and 30% in urine and blood, respectively. A total of six proficiency testing samples
(UKNEQAS 2003, Labquality 2004) containing buprenorphine were analyzed with the established method,
and the results were within II% of the consensus or average value.
Discussion: The present method has been applied to blood and urine samples from more than 420 autopsy
cases and 670 living patients. Interferences from sample matrix have been detected only very rarely even
though the surviving parent ions are monitored to obtain adequate selectivity. Successful participation in
international proficiency testing schemes proves the high reliability ofthe method.
Keywords: Buprenorphine, Quantification, LC-MS/MS
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