Urinary Excretion Rates of Ketamine and Norketamine Following ...

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Journal of Analytical Toxicology, Vol. 28, July/August 2005 dreamy thinking, disturbances of speech, hearing and seeing, lack of muscle coordination, disorientation, anxiety, disinhibi- tions, euphoria, seeing the world differently, and irrational be- havior. Higher doses cause great difficulty in moving, respiratory disturbances, seizures, and nausea. Extreme doses produce com- plete disassociation from reality and loss of consciousness, hal- lucinations, out-of-body experiences, and so-called "near-death experiences". Frequent intake of ketamine can lead to psycho- logical and physical dependency and tolerance (12-14). Deaths by overdose in the absence of other drugs are excep- tionally rare (5,15). Overdose in the case of ketamine is a very relative term, because doses used by drug-addicts are typically lower than necessary for anesthesia. The highest therapeutic dose is 13 mg/kg, while doses taken to induce psychedelic effects rarely exceed 2 mg/kg. Accidental deaths such as falling from a great height, death by hypothermia, car accident, or drowning in a bathtub are the most common ketamine-related deaths (7). Ketamine is rapidly metabolized to an active metabolite, norketamine, and an inactive metabolite, 6-hydroxynorketamine. The major pathway is N-demethylation to norketamine by the microsomal cytochrome P450 system (8). Norketamine is also hydroxylated to 6-hydroxynorketamine, conjugated, and ex- creted in the urine. Hydroxylation and conjugation of glu- curonic acid with norketamine and its hydroxylated metabolites generate water-soluble compounds to facilitate urinary excretion (9). Norketamine also undergoes dehydrogenation to dehydronorketamine. It had been suggested that dehydronorke- Table I Urinary Excretion of Ketamine and Norketamine Following Ketamine Administration to Children* Concentrations (ng/mL) Measured by Concentrations (ng/mL) Measured by LC-MS-APCI GC-MS-NCI LC-MS-APCI GC-MS-NCI Day Ketamine Norketamine Ketamine Norketamine Day Ketamine Norketamine Ketamine Norketamine Case 1 0-1 urine samples not collected 2 - 50 - 57 3 - 7 - 3 4 - 5 - 2 5 - 5 - 0.4 6 - 4 - 0.4 7 - - 0.2 8 - - 0.06 9 - - 0.1 10 - - 0.2 11 - - 0.2 12 - - - 0.1 13 - - 0.08 14 - - - 0.05 15-16 urine samples tested negative Case 2A 0 urine sample not collected 1 41 264 41 227 2 4 18 - 11 3 - 3 - 2 4 - - - 0.4 5 - - - 0.6 Case 2B 0 799 409 695 534 1 52 387 58 325 3 3 2 - 2 5 - - - 0.1 7 2 - - - 9 6 - - - 11 4 - - - Case 2C 0 urine sample not collected 1 813 548 1181 743 2 195 417 296 3 17 85 - 4 7 17 - 5 3 6 - Case 3 0 urine sample not collected 1 161 527 2 110 34 3 - 5 4 - 3 5 - 2 6 7 Case 4 0 502 1276 1 12 206 2 - 5 3 - 2 5 - 3 6 7-15 urine samples tested negative Case 5 179 156 586 496 0 urine sample not collected 1 30 166 29 147 Case 6 82 13 3 430 52 2 2 1182 0 1204 1559 1410 1442 0.7 0.1 184 1 1 467 535 642 553 2 55 52 71 72 3 - 6 - 3 4 - 4 - 1 5 - 3 - 0.6 6 - - 0.1 * Day 0: the day of ketamine administration; Day 1 : the first day after ketamine administration; Cases 2A, 2B, and 2C represent urine samples collected from one patient to whom ketamine was administered three times during two-year period. 0.06 0.6 0.1 0.07 377
tamine was an artifact of the analytical procedure [using a gas chromatography (GC) technique[ rather than a metabolite (16). Other authors (17) have detected ketamine, norketamine, and dehydronorketamine in urine by liquid chromatography-mass spectrometry (LC-MS). Norketamine has one-third the activity of ketamine (18). Since the early 1990s, interest in this substance has grown among young people and an increasing number of reports have appeared about recreational use of this drug (19-22). Ketamine is a "club drug" and its illicit use by teenagers at rave parties has also been reported. The major source of ketamine is diversion from veterinary clinics. It is also smuggled from countries where it is not legally controlled. Due to complex synthesis and difficulties with buying precursors, solvents, and reagents, ketamine is not manufactured in clandestine laboratories. In pharmaceutical preparations, ketamine is sold in liquid form and the powder form is prepared by boiling off the solution (18). It can be taken via intramuscular, intravenous, intranasal, oral, and rectal routes. Ketamine can also be smoked, some- times in combination with marijuana or tobacco (5,23). Oral doses are prepared from the powder by dissolving it in water, juice, or alcoholic beverages. Recently it has also been identified as a "date-rape" drug used for drugging unsuspecting victims and then raping them while the victim is still under its influence. In a typical scenario, the perpetrator surreptitiously adds ketamine to the alcoholic bev- erage of an unsuspecting person, who is subsequently sexually assaulted while under the influence of this substance. Many victims do not report the incident until several days after the event (24-26). These situations create a demand for sensitive analytical methods to reveal the presence of the drug and/or metabolites in biological specimens collected from the subject. An important parameter in drug testing for forensic purposes is the detection window (i.e., how long after drug administration a person tests positive for the drug or metabolite). Obviously, detection times are highly dependent upon a number of factors including dosage, individual rates of metabolism and excretion, method specificity, and detection limits. Consequently, detection times may vary between methods, and it is important to characterize the performance of new methods with clinical drug specimens obtained under controlled conditions. Many analytical methods for the determination of ketamine and norketamine in biological materials are described in the lit- erature. Initially, conventional (27) and two-dimensional (28) thin-layer chromatography methods were applied, but these methods require high specimen volume and often lack the re- quired sensitivity. GC with nitrogen-phosphorus detection or electron capture detection methods, have been used for bio- transformation and disposition studies. However, validation pa- rameters were not defined or limits of detection (LOD) ranged from 10 to 100 ng/mL, which is insufficient. Many authors developed HPLC with UV detection methods for separation, detection, and quantification of ketamine and norketamine, which can attain LODs of 5 ng/mL (29-32). Only GC-MS or LC-MS methods provide the sensitivity, selectivity, and specificity nec- 378 Journal of Analytical Toxicology, Vol. 28, July/August 2005 essary for qualitative and quantitative data needed in forensic studies on a single ketamine exposure used as the date rape drug. These methods have been developed and described recently (17,33). This paper describes two analytical methods, GC-MS-negative chemical ionization (NCI) and LC-MS-atmospheric pressure chemical ionization (APCI), and their application to the analysis of urine samples collected from hospitalized children who received ketamine as an anesthetic. The GC-MS-NCI method was published previously by Negrusz et al. (34) and the authors of this article developed the LC-MS-APCI method. Materials and Methods Chemicals Ketamine, norketamine, ketamine-d4, and norketamine-d4 were purchased from Cerilliant Corporation (LGC Promochem, Warszawa, Poland). The enzyme [3-glucuronidase (Type H-2 crude solution, 110,350 units/mL from Helix pomatia) and heptafluorobutyric anhydride (HFBA) were acquired from Sigma-Aldrich (Poznan, Poland). The HCX Isolute | (10-mL, 200-rag) extraction columns (product of International Sorbent Technology, Hengoed, U.K.) were supplied by Able & Jasco (Krakow, Poland). All solvents and reagents were of analytical grade. Biological material Urine samples were collected from six hospitalized children (age 4-13 years, weight 17-68 kg) who had received a single intravenous dose of ketamine as an anesthetic for short surgical procedures. One child received ketamine three times during a two-year period (case 2A, 2B, and 2C, Table I). The doses ranged from 0.75 to 1.59 mg/kg. Individual urine samples were collected every day or once every two days for 4-16 days. If the urine samples were not analyzed immediately, they were stored up to one month at-18~ prior to analysis. For validation of the LC-MS-APCI method, control urine samples were taken from healthy persons with no history of ketamine use. Solid-phase extraction (SPE) To the urine samples (2 mL for GC-MS-NCI procedure or 1 mL for LC-MS-APCI procedure), an appropriate internal standard (IS, 1 ng of norketamine-d4 in 10 tJL of methanol for the GC-MS-NCI method and 100 ng each of norketamine-d4 and ketamine-d4 in 10 IlL of methanol, for the LC-MS-APCI method) was added. This was followed by the addition of 1 mL of 0.1M acetate buffer (pH 5.5) and 50 IJL of ~-glucuronidase before the specimens were incubated for 90 min at 37~ After this, 1 mL of 1.93M acetic acid and deionized water (10 mL for GC-MS-NCI or 5 mL for LC-MS) were added to the samples and applied on the mixed-mode Isolute HCX SPE columns. Columns were preconditioned with methanol (3 mL), deionized water (3 mL), and 1.93M acetic acid (1 mL). The pretreated sample was slowly passed through the column
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