Urinary Excretion Rates of Ketamine and Norketamine Following ...

Journal of Analytical Toxicology, VoL 28, July/August 2005
(approximately 5 rain) followed by rinsing with 3 mL of deion-
ized water, 1 mL of 0.1M HC1, and 3 mL of methanol. The
column was dried under vacuum for 5 rain. The retained ana-
lytes were eluted with a mixture of methylene chloride/iso-
propanol/ammonium hydroxide (78:20:2, v/v) under gravity.
The eluates were evaporated to dryness under a stream of air. In
cases of analyte concentrations exceeding the upper level of
the respective calibration curve, the sample was diluted and the
analysis was repeated.
GC-MS-NCI analysis
The dry residues were derivatized with 50 1JL of HFBA at
60~ for 30 rain. After incubating, the excess derivatizing
reagent was evaporated under air and the dry residue reconsti-
tuted in 50 I~L of ethyl acetate before I IJL was injected into the
GC-MS system.
Quantitation of ketamine and norketamine was carried out
using the Agilent Technologies 6890 series GC equipped with a
5973 series mass selective detector (MSD) and a 6890 series au-
tosampler (Agilent Technologies, Wilmington). The NCI mode
was applied using methane as the reagent gas. The separation
of analytes was performed with the use of a HP-5MS fused silica
capillary column (30-m 0.25-mm i.d., 0.25-1Jm film thick-
ness). The capillary inlet system was operated in the splitless
mode. Instrumental conditions were as follows: injection port,
280~ GC temperature program, 60~ for 1 min, ramp to
310~ at 30~ and hold 3 min; transfer line, 280~ source,
200~ quadrupole, 230~ The flow rate of carrier gas (helium)
was I mL/min. The quantitative (underlined) and qualifier ions
monitored for each derivatized compound were ketamine, m/z
226, 357; norketamine, m/z 383 and 399; and norketamine-d4,
m/z 387 and 403.
LC-MS-APCI analysis
The dry residue (after SPE and without derivatization) was re-
constituted in 100 lJL of an acetonitrile (ACN)/water mix (1:4
v/v). A 20-1~L sample was injected by autosampler into the
LC-MS system.
Analysis was performed with the HP-1100 series LC coupled
to a MS equipped with an APCI interface (Hewlett Packard,
Wilmington).
The separation of analytes was performed with the use of a
LiChroCART Purospher STAR RP-18e column (55- 4-mm
i.d.) (Merck, Darmstadt, Germany) thermostated at 25~ The
mobile phase consisted of 0.1% (v/v) formic acid in water and
ACN. The flow rate was 0.8 mL/min. All analyses were carried
out in gradient mode: 0 min-10% ACN, 5 min-40% ACN, 6
min-10% ACN, and 8 min-10% ACN.
Nitrogen generated by a Whatman apparatus was used as a
nebulizing gas. First, an autotuning procedure was carried out.
Then optimization of MS parameters was accomplished by flow
injection analysis to obtain the most intense ions for selected
ion monitoring mode. Ketamine and norketamine at concen-
trations of 100 ng/mL in mobile phase were individually injected
directly into the MSD without chromatographic separation and
analyzed in full-scan mode (m/z range 50-500 ainu). For both
analytes, the pseudomolecular ions of ketamine (m/z 238) and
of norketamine (m/z 224) were selected as the most repro-
ducible and intense. Other parameters were as follows: frag-
mentor voltage, 60 V; vaporizer temperature, 330~ capillary
voltage, 4200 V; drying gas flow, 7 L/rain; temperature, 300~
nebulizer pressure, 35 psi; corona current, 4.5 IIA. All data were
acquired and analyzed by HP software, ChemStation version
A.06.03 for Windows NT.
The calibration curves for ketamine and norketamine in urine
consisted of 11 points for each compound, which covered the
range of 0.5-2000 ng/mL. The calibrator solutions were pre-
pared by adding known amounts each of the IS (ketamine-d4
and norketamine-d4, 100 ng/mL each), ketamine, and norke-
tamine to control urine (0, 0.5, 1, 2, 5, 10, 20, 100, 500, 1000,
and 2000 ng/mL). The samples were hydrolyzed, extracted, and
analyzed by LC-MS-APCI in the same manner as described for
the patients' urine samples. Peak-area ratios (ketamine/ke-
tamine-d4; m/z 238/242 and norketamine/norketamine-d4; m/z
224/228) were calculated for each standard and plotted against
the known concentration of the standard.
The method was validated by repetitive (at least three times)
analysis of two different concentrations of spiked control urine
samples containing 40 and 750 ng/mL of both target analytes
(on the same day and over a period of three weeks).
LOD, lower limit of quantification (LLOQ), and limit of lin-
earity (LOL), as well as extraction recovery for ketamine and
norketamine (at concentrations of 40 and 750 ng/mL from
spiked urine in comparison with unextracted drug solutions)
were determined. Calculations were performed using Merck's
Validation Manager Program.
Results
The GC-MS-NCI method (34) had an LOQ of 20 ng/mL for ke-
tamine and 0.05 ng/mL for norketamine, and displayed a LOL
across a concentration range of 20-1000 ng/mL and 0.050-1500
ng/mL, respectively.
The LC-MS-APCI method was characterized by the same val-
idation parameters for both compounds: LOD of 0.5 ng/mL,
LLOQ of 2 ng/mL, and LOL of 2-2000 ng/mL. Linear regression
correlation coefficients of the calibration curves were 0.9997 for
ketamine and 0.9999 for norketamine. Recovery of ketamine
from urine was 107.86 3.66% at 40 ng/mL and 100.33
1.87% at 750 ng/mL. Recovery for norketamine at the same low
and high concentrations was 101.18 3.69% and 100.59
1.75%, respectively. All values were high, but comparable and
reproducible. Coefficient of variations of the intra- and in-
terassay precision did not exceed 4% for ketamine and norke-
tamine at low concentration (40 ng/mL). These data for both an-
alytes at the 750-ng/mL concentration were slightly lower. For
ketamine the coefficient of variation was less than 2%.
On the basis of the successful validation, both methods were
applied to the determination of ketamine and norketamine in 62
urine samples collected from six hospitalized children. Urinary
excretion profiles of ketamine and norketamine were presented
in Figures 2-5 and Table I.
After a single intramuscular ketamine dose, the drug was de-
tected in the urine samples of five children. By the GC-MS-NCI
379