SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A83
DETERMINA TION OF NALOXONE AND NORNALOXONE IN HUMAN PLASMA BY HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY - ELECTROSPRAY IONIZATION ­
TANDEM MASS SPECTROMETRY
Wenfang B. Fang' and David E. Moody, Center for Human Toxicology, Department of Pharmacology and
Toxicology, University ofUtah, Salt Lake City, UT 84112
Naloxone is a jl-opioid antagonist that has been used clinically for over forty years mainly for the treatment
of opioid overdose and to reverse narcotic-induced depression following surgery. A more recent
application is the use of naloxone in a combination tablet containing buprenorphine at a fixed ratio of 4: I
(buprenorphine: naloxone) for the treatment of opioid dependence. The purpose of the addition of
naloxone to buprenorphine tablet is to prevent diversion ofbuprenorphine for illicit Lv. use. ~ecause ofthe
low doses of naloxone used in maintenance therapy, sensitive analytical methods are required. This has
traditionally proven difficult for 6-keto nor-opiates such as nornaloxone. A highly sensitive method was
developed to measure naloxone and its metabolite nornaloxone in human plasma to gain further knowledge
about their metabolism and pharmacokinetics. drNaltrexone and d 3 -oxymorphone were used as internal
standards for naloxone and nornaloxone. Preliminary experiments demonstrated that solid-phase extraction
improved the recovery ofnorna lox one to 30% compared to 10% using liquid-liquid extraction. Solid-phase
extraction was applied for sample preparation using C18 extraction columns and 0.01 M ammonium
carbonate buffer (pH = 9). High performance liquid chromatography interfaced by electrospray ionization
to a tandem mass spectrometric detector (HPLC-ESI-MSIMS) was used for quantitation. A XTerra MS
CI8 3.5 Jlm 2.1x 50 mm column (Waters Corporation, Milford, MA) was used for separation. The mass
spectrometer was a Finnigan model TSQ7000 Thermo Quest triple-stage quadrupole. Quadrupole 1 was
set to pass only ions at m/z 328, 288, 345 and 305 that correspond to the MH+ ions of naloxone,
nornaloxone and their internal standard d 3 -naltrexone and d 3 -oxymorphone. The MH+ ions were caused to
undergo collision induced dissociation in quadrupole 2 that produced product ions at mlz 310, 270,327 and
287 respectively, which were then monitored selectively by quadrupole 3. The calibration range was from
0.075 to 20 ng/mL for naloxone and 1 to 20 ng/ml for nornaloxone with the calibration curve constructed as
quadratic with llX weighting. The lower limit of quantitation (LLOQ) was determined at 0.075 ng/ml for
naloxone and 1 ng/ml for nornaloxone. Specificity for naloxone and nornaloxone was determined from
analysis of blank plasma fortified with internal standard only (3 replicates) and with LLOQ concentration
(1 replicate) in six different lots of plasma. The primary evaluation was to compare the mean peak area
ratio of any signal at the retention time of naloxone and nornaloxone to its internal standard for each lot
with the mean peak area ratio of the six LLOQ samples. Mean ratios relative to mean LLOQ ranged from
2.68 to 7.72 with a mean of 5.35% for naloxone and from 8.03 to 25.5 with a mean of 16.5% for
nornaloxone. Intra-run accuracy of the LLOQ was within 5.1 % of target with intra-run precision within
13.5% for naloxone and within 12% of target and with intra-ran precision within 7.4% for nornaloxone.
(Supported by NIDA grant ROIDAIOIOO)
Keywords: Naloxone, Nornaloxone, HPLC-ESI-MS/MS
Page 197