Urinary Excretion Rates of Ketamine and Norketamine Following ...

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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 analytes 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 autosampler (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 concentrations 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 reproducible 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 norketamine 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/ketamine-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 ketamine 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 validation 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 norketamine at low concentration (40 ng/mL). These data for both analytes 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 detected in the urine samples of five children. By the GC-MS-NCI 379
method, ketamine was determined in the urine of two children up to i day after exposure in concentrations ranging from 41 to 695 ng/mL; in the next two children up to 2 days after drug administration in concentrations ranging from 71 to 1410 ng/mL, and in one child only on the day of drug administration, when its concentration was 586 ng/mL. Norketamine was detected in the urine of two children up to 6 days after dosing (in concentrations ranging from 0.07 ng/mL to 1442 ng/mL), in one child up to 5 days (in concentrations of 0.6-227 ng/mL), in another child up to 7 days (0.06-430 ng/mL), and in the last child up to 14 days (0.05-57 ng/mL). Using the LC-MS-APCI method, ketamine was detected (at concentrations of 4-1204 ng/mL) in three children up to two days after exposure and in two children up to one day (at concentrations of 12-502 ng/mL) after exposure. Norketamine was detected up to three (at concentrations of 2-409 ng/mL), five (2-1559 ng/mL), and six days (4-50 ng/mL) after drug administration. Using either method, neither ketamine nor norketamine were detected in the urine taken from one child through the entire 16-day period. Data from Case 2 (Table I) demonstrated that repeated doses of ketamine (three times during a two-year period) resulted in its slower elimination. After the second dose (case 2B), the elimination of parent ketamine was extended to 11 days, and after the third dose it was extended to 5 days after drug admin- 2 mc~e ! iC~se 2A DC~se 2B , ~ Case 2C i Case 3 ! Case 4 i I 9 Case 5 [ [] Case 6 Figure 2. The urinary excretion profiles of ketamine for six hospitalized children determined by GC-MS-NCI. 2 i RCase 1 : 9 Case 2A DCase 2B D Ca-se 2C it Case 3 m Case 4 [lCase 5 1 [oca.se 6 [ Figure 3. The urinary excretion profiles of norketamine for six hospitalized children determined by GC-MS-NCI. 380 Journal of Analytical Toxicology, Vol. 28, July/August 2005 istration. After each dose norketamine was excreted in the same 5-day period. Overall elimination times of ketamine varied between the different children and the different methods. Discussion Because ketamine has become a popular club drug and date- rape drug, it is a subject of interest to forensic toxicologists, es- pecially regarding its detection times. Detection times indicate how long after drug administration a person excretes a drug or metabolite at a concentration above a specific method LOD. This makes sensitive methods with low LODs essential. The methods used in this study allowed for quantification of ketamine and norketamine at very low concentrations. The GC-MS-NCI method had an LOQ of 20 ng/mL for ketamine and 0.05 ng/mL for norketamine, and the LC-MS-APCI method provided an LOQ of 2 ng/mL for both compounds. The applied LC-MS-APCI method is more sensitive than elaborated by Moore et al. (17) who measured ketamine and norketamine with a LOD of 4 ng/mL for both analytes. A previously reported GC-MS method achieved an LOQ for ketamine of 13 ng/mL and an LOQ for norketamine of 9 ng/mL (35). Both applied methods have advantages and disadvantages. ~2 = 9 Case I iCase 2A []Case 2B [Case 2C i 9 Case 3 D Case 4 9 Case 5 [] Case 6 Figure 4. The urinary excretion profiles of ketamine for six hospitalized children determined by LC-MS-APCI. BCase 1 9 C~tse 2A DC~se 213 DC~se 2C 9 Case 3 9 Case 4 9 Case 5 DC~se 6 Figure 5. The urinary excretion profiles of norketamine for six hospitalized children determined by LC-MS-APCl.
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