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Analysis of ketamine and norketamine in urine by automatic solid-phase extraction (SPE) and positive


Forensic Science International 174 (2008) 197–202 www.elsevier.com/locate/forsciint

Analysis of ketamine and norketamine in urine by automatic solid-phase extraction (SPE) and positive ion chemical ionization–gas chromatography–mass spectrometry (PCI–GC–MS)
Eun-mi Kim *, Ju-seon Lee, Sang-kil Choi, Mi-ae Lim, Hee-sun Chung
Narcotics Analysis Section, National Institute of Scienti?c Investigation, 331-1 Shinwol 7-dong, Yangcheon-gu, Seoul 158-707, Republic of Korea Received 16 April 2007; accepted 23 April 2007 Available online 5 June 2007

Abstract Ketamine (KT) is widely abused for hallucination and also misused as a ‘‘date-rape’’ drug in recent years. An analytical method using positive ion chemical ionization–gas chromatography–mass spectrometry (PCI–GC–MS) with an automatic solid-phase extraction (SPE) apparatus was studied for the determination of KT and its major metabolite, norketamine (NK), in urine. Six ketamine suspected urine samples were provided by the police. For the research of KT metabolism, KTwas administered to SD rats by i.p. at a single dose of 5, 10 and 20 mg/kg, respectively, and urine samples were collected 24, 48 and 72 h after administration. For the detection of KT and NK, urine samples were extracted on an automatic SPE apparatus (RapidTrace, Zymark) with mixed mode type cartridge, Drug-CleanTM (200 mg, Alltech). The identi?cation of KT and NK was by PCI–GC–MS. m/z 238 (M + 1), 220 for KT, m/z 224 (M + 1), 207 for NK and m/z 307 (M + 1) for Cocaine-D3 as internal standard were extracted from the full-scan mass spectrum and the underlined ions were used for quantitation. Extracted calibration curves were linear from 50 to 1000 ng/mL for KT and NK with correlation coef?cients exceeding 0.99. The limit of detection (LOD) was 25 ng/mL for KTand NK. The limit of quantitation (LOQ) was 50 ng/mL for KT and NK. The recoveries of KT and NK at three different concentrations (86, 430 and 860 ng/mL) were 53.1 to 79.7% and 45.7 to 83.0%, respectively. The intra- and inter-day run precisions (CV) for KTand NK were less than 15.0%, and the accuracies (bias) for KTand NK were also less than 15% at the three different concentration levels (86, 430 and 860 ng/mL). The analytical method was also applied to real six KT suspected urine specimens and KTadministered rat urines, and the concentrations of KTand NK were determined. Dehydronorketamine (DHNK) was also con?rmed in these urine samples, however the concentration of DHNK was not calculated. SPE is simple, and needs less organic solvent than liquid–liquid extraction (LLE), and PCI–GC–MS can offer both qualitative and quantitative information for urinalysis of KT in forensic analysis. # 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Urinalysis of Ketamine; SPE; PCI–GC–MS

1. Introduction Ketamine (KT) is a rapid-acting anesthetic used during surgical procedures in both animals and humans under the name ‘‘Ketalar’’ [1]. The chemical structure, mechanism of action and pharmacological effects are similar to those of phencyclidine (PCP), but KT is much less potent than PCP. Since KT is odorless and tasteless, it can be added to beverages, without being detected, to induce amnesia. Because of such properties, the drug is sometimes misused in a sexual assault, referred to as date-rape drug.

* Corresponding author. Tel.: +82 2 2600 4931; fax: +82 2 2600 4939. E-mail address: emkim@nisi.go.kr (E.-m. Kim). 0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.04.217

KT is metabolized to at least two compounds: ?rst, by Ndemethylation, to norketamine (NK), which has 1/3–1/2 of the potency of KT. NK is further dehydrogenated to produce dehydronorketamine (DHNK) [1,3]. The parent compound and both major metabolites are further transformed by hydroxylation and conjugation prior to elimination (Fig. 1). About 90% of a dose is excreted in the urine in 72 h, with about 2% of the dose as unchanged drug, 2% as NK, 16% as DHNK and 80% as conjugates of hydroxylated metabolites [4]. For hallucination, it is supplied as 200–525 mg of KT for oral administration, 50–70 mg for intra-muscular injection or 100–250 mg for inhalation and rectal administration [1,2]. Even though no fatal cases from KT overdose have been reported in Korea, the use of KT by drug-rape criminals is increasing in this society. As a result, the Korean

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E.-m. Kim et al. / Forensic Science International 174 (2008) 197–202 single dose of 5, 10 and 20 mg/kg, and urine samples were collected for 24, 48 and 72 h after administration. Four rats per each group were used. Six drug abuser’s urine samples from the police, which were positive for KT, were collected for quantitative analysis. All urine samples were centrifuged and supernatants were stored at ?20 8C until analysis.

2.3. Sample preparation
Urine extraction was performed on automatic SPE equipment, RapidTraceTM, Zymark Co., USA. The SPE column, Drug-CleanTM C (200 mg), was purchased from Alltech Co., USA. Column were preconditioned by adding 2 mL methanol and 2 mL distilled water followed by the addition of 30 mL 5NHCl and 50 mL internal standard. Urine samples (made up to 3 mL with distilled water) were loaded on to columns at a rate of 2 mL/min. Column were washed with 2 mL 0.1 N-NaOH, 2 mL distilled water and 4 mL hexane in sequence. KT, NT and internal standard were eluted with 3 mL methylene chloride/ isopropanol (25:75). The eluates were evaporated under a gentle stream of nitrogen. Residues were reconstituted in 50 mL ethylacetate and transferred to autosampler vials containing reduced volume inserts.

Fig. 1. Metabolic pathways of ketamine [3].

Government has classi?ed it as a controlled drug since November 2005. In this study, we established an analytical method for KT and NK in urine samples using solid-phase extraction (SPE) and positive ion chemical ionisation–gas chromatography–mass spectrometry (PCI–GC–MS). We identi?ed and quanti?ed KT and its metabolites in animal urine, and in the urine of six drug abusers.
2. Experimental 2.1. Chemicals and reagents
Ketamine hydrochloride (as free base; 1 mg/mL in methanol), norketamine hydrochloride (as free base; 1mg/mL in methanol) and cocaine-D3 (100 mg/mL in methanol) were purchased from Cerilliant Co., USA. The mixture of ketamine (KT) and norketamine (NK) (100 mg/mL in methanol) standard stock solution was diluted with methanol to appropriate concentrations as needed. The internal standard, cocaine-D3 was diluted to 4 mg/mL with methanol. All other chemicals and solvents were of analytical grade.

2.4. PCI–GC–MS analysis
An Agilent GC–MS instrument (6890N GC and 5975 MSD) operating in chemical ionization mode was used for the analysis. Methane was used as reagent gas and an Agilent HP-5MS capillary column (30 m ? 0.2 mm i.d., 0.25 mm ?lm thickness) was used for separation. The GC was operated in splitless mode and the injection volume was 1 mL with an Agilent 7683 autosampler. The injector temperature was 265 8C, the transfer line was 270 8C, and the source and quadrupole were kept at 230 and 150 8C, respectively. The initial GC oven temperature of 100 8C was held for 1 min, and then increased at a rate of 15 8C/min until the ?nal temperature of 280 8C was attained. The ?nal temperature was held for 5 min. For identi?cation, the following ions were monitored from full-scan mass spectrum: m/z 238 (M + 1), 220 for KT, m/z 224 (M + 1), 207 for NK and m/z 307 (M + 1) for cocaine-D3, internal standard (the underlined ions are used for quantitation).

2.5. Validation of the method
The validation of the method was carried out by establishing linearity, intraand inter-assay accuracy and precision, limit of detection (LOD) and quantitation (LOQ), recovery. The standard curves were made by spiking blank urine samples with corresponding analytical working solutions to obtain calibration concentrations 50, 100, 200, 500 and 1000 ng/mL of KT and NK. Inter- and intra-assay accuracy and precision data for KT and NK were determined with

2.2. Animal, human subjects, drug administration and urine sampling
Male Sprague–Dawley rats (wt. 180–200 g) were used and kept in individual metabolic cages. Three groups of rats were administered (i.p.) with KT at a

Fig. 2. Total ion chromatogram of ketamine and norketamine spiked urine.

E.-m. Kim et al. / Forensic Science International 174 (2008) 197–202 low (86 ng/mL), medium (430 ng/mL) and high (860 ng/mL) QC samples. The absolute recovery was determined at three concentrations (86, 430 and 860 ng/mL).

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3. Results and discussion 3.1. Analysis of KT and NK by PCI–GC–MS The extracts obtained from SPE were analyzed by PCI–GC– MS. The total ion chromatogram of spiked standard solutions and internal standard was shown in Fig. 2. These drugs were well separated at retention time of 9.81 for KT, 9.51 for NK and 12.07 min for internal standard (Fig. 2). Mass spectra of KT and NK were shown in Figs. 3 and 4. As the result of proton transfer, [M+H]+, pseudomolecular ion, m/z 238 for KT and m/z 224 for NK were determined, respectively. 3.2. Analytical method validation results The parameters of the validated method for analysis of KT and NK are shown in Table 1. Extracted calibration curve of KT and NK were linear from 50 to 1000 ng/mL with correlation coef?cient 0.99. The limit of

quantitation of KT and NK was the ?rst point of the calibration curve, 50 ng/mL, and the limits of detection, which were de?ned as the detection limits of the target and quali?er ion peaks on each mass chromatogram (S/N = 3), was estimated to be 25 ng/mL in the full-scan modes. The values of accuracy for KT and NK at three different concentration levels were less than 15% (20% at low QC) except for KT at the medium QC, 21.54%. Precision was calculated by performing a one-way ANOVA test [5], the values of within-run precision for KT and NK were less than 15%, and the values of between-run precision were less than 10% at three different levels. The average absolute recovery for KT and NK was 70.1 and 64.5%, respectively. 3.3. Identi?cation and quantitation of KT and its metabolites in rat urine Three doses of KT (5, 10 and 20 mg/kg) were administered to rat by i.p. and urines were collected for 24, 48 and 72 h after administration. Fig. 5 shows an example of a total ion chromatogram by PCI–GC–MS for 24 h urine after 20 mg/kg KT administration. KT and its metabolites, NK and dehydronorketamine (DHNK) were identi?ed at the retention time

Fig. 3. PCI-mass spectrum of ketamine (KT).

Fig. 4. PCI-mass spectrum of norketamine (NK).

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E.-m. Kim et al. / Forensic Science International 174 (2008) 197–202

Table 1 Validation data of ketamine and norketamine for the established procedure in urine Parameters Linearity range (ng/mL) LOD (ng/mL) LOQ (ng/mL) Accuracy (%) At low QC (86 ng/mL) At medium QC (430 ng/mL) At high QC (860 ng/mL) Precision (%) at low QC Intra-day Inter-day Precision (%) at medium QC Intra-day Inter-day Precision (%) at high QC Intra-day Inter-day Recovery (%) Low QC Medium QC High QC KT Y = 0.0028x ? 0.17 (r2 = 0.9948) 25 50 15.90 ?21.54 5.59 3.73 1.66 4.48 1.44 14.38 5.30 79.7 77.6 53.1 NK Y = 0.0068x ? 0.3723 (r2 = 0.9921) 25 50 2.54 ?2.10 ?2.33 4.83 0.49 9.33 7.01 11.15 3.82 83.0 64.9 45.7

of 9.84, 9.61 and 9.78 min, respectively (Fig. 5). The PCI-mass spectrum of DHNK was shown in Fig. 6. As the result of a proton transfer to DHNK molecule (MW 221), m/z 222 of [M+H]+ was identi?ed. In the 24 h urine after KT administration, the concentrations of KT and NK were increased in a dose-dependent way (Table 2), and the average urinary metabolic ratio of NK to KT for 5, 10 and 20 mg/kg KT administration was 1.90, 9.33 and 9.39, respectively. The concentration of NK was higher than KT in all KT administration groups (Table 2). Even though the concentration of DHNK was not calculated in this study because of a lack of standard material, when it was calculated as the ratio to internal standard, the amount of metabolites was in the order of NK > DHNK > KT in 24 h urine. Meanwhile traces of NK were detected in the 48 h urine, and there were no KT and NK detected in 72 h urine (data not shown). 3.4. Concentrations of KT and NK in drug abuser’s urines The concentration of KT and NK in 6 KT suspected urines were in the range 0.03–56.16 and 0.42–29.31 mg/mL, respectively (Table 3). The ratio of NK to KT ranged from 0.28 to 2.04. Fig. 7 shows an example of the total ion chromatogram for a KT abuser urine by PCI–GC–MS. Wieber et al. [6] reported that over a 72 h period a single dose of KT was eliminated primary in the urine as unchanged drug (2.3%), NK (1.6%), DHNK (16.2%) and conjugates of hydroxylated derivatives of KT (80%). Even though the accurate concentration of DHNK was not calculated in this study, our results showed similar pattern with Wieber’s research. When the ratios of DHNK to internal standard were compared to the ratio of NK, the excretion amount of DHNK was more than 8–38 times (average 15.6 times) of NK in KT abuser urines (data not shown). Lin and Lua [7] also reported that the DHNK concentration was greatest in 20 of the 24 KT positive urine samples. The concentration of DHNK will be studied with authentic DHNK material in the future.

Standard curves were analyzed in blank urine samples (1 mL) spiked with different concentrations of analytes (50, 100, 200, 400, 800 and 1000 ng) (n = 5). For estimation of accuracy and precision 3–5 urine samples which were spiked with three different concentrations (86, 430 and 860 ng/mL) of KT and NK were extracted and analyzed by PCI–GC–MS. The extraction procedure was repeated independently on three successive days. The value within ?15% (?20% at low QC) were deemed acceptable. For recovery, urine samples, which were spiked three different concentrations were extracted by SPE and calculated as concentration corresponding to aliquot of QC solutions in methanol (n = 3). Internal standard was added in the last step of extraction procedure and injected to GC–MS.

Fig. 5. Total ion chromatogram of rat urine for 24 h after ketamine administration (20 mg/kg, i.p.).

E.-m. Kim et al. / Forensic Science International 174 (2008) 197–202

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Fig. 6. PCI-mass spectrum of dehydronorketamine (DHNK).

Table 2 Urinary concentrations of ketamine and norketamine for 24 h after ketamine administration in rat Dose (mg/kg) 5 n 1 2 3 4 M ? S.D. 10 1 2 3 4 M ? S.D. 20 1 2 3 4 M ? S.D. KT (ng/mL) 479.23 1000.08 440.54 728.19 662.01 ? 258.93 2995.18 1226.51 1688.33 2310.78 2055.20 ? 768.16 6814.47 4433.57 2097.07 1993.49 3834.65 ? 2283.79 NK (ng/mL) 1031.26 1444.83 1014.31 1245.21 1183.90 ? 203.23 19328.33 11493.35 21026.28 20885.33 18183.32 ? 4525.85 55607.92 37749.77 28011.34 15036.46 34101.37 ? 17091.93 Ratio of NK/KT 2.15 1.44 2.33 1.71 1.90 ? 0.40 6.45 9.37 12.45 9.04 9.33 ? 2.46 8.16 8.51 13.36 7.54 9.39 ? 2.67

Urine samples were diluted to 10–100 times when the concentration was higher than 1000 ng/mL.

Fig. 7. Total ion chromatogram of a ketamine positive human urine.

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Table 3 Concentrations of ketamine and norketamine in six ketamine positive human urines Sample 1 2 3 4 5 6 KT (mg/mL) 0.78 56.16 37.03 18.53 0.22 0.03 NK (mg/mL) 1.60 29.31 15.87 5.20 0.42 nd Ratio of NK/KT 2.04 0.52 0.43 0.28 1.86 –

concentrations will be investigated in further studies. SPE is simple, and requires less organic solvent than liquid–liquid extraction (LLE), and PCI–GC–MS can offer both qualitative and quantitative information for urinalysis of KT in the forensic science. References
[1] A. Mozayani, Ketamine-effects on human performance and behavior, Forensic Sci. Rev. 14 (2002) 123–131. [2] E. Tanaka, K. Honda, H. Yasuhara, Ketamine: its pharmacology and toxicology, HOUJUDOK 23 (2005) 187–191. [3] R.C. Baselt, Disposition of Toxic Drugs and Chemicals in Man, seventh ed., Chemical Toxicology Institute, Foster city, CA, 2004. [4] A.C. Moffat, M.D. Osselton, B. Widdop, Clarke’s Analysis of Drugs and Poisons, third ed., Pharmaceutical Press, London, 2004. [5] F.T. Peters, Analytical Method Development and Validation FBI Laboratory Symposium on Forensic Toxicology, August 29–30, Washington, DC, USA, 2004. [6] J. Wieber, R. Gugler, J.H. Hengstmann, H.J. Dengler, Pharmacokinetics of ketamine in man, Anaesthesia 24 (1975) 260–263. [7] H.R. Lin, A.C. Lua, Detection of acid-labile conjugates of ketamine and its metabolites in urine samples collected from Pub participants, J. Anal. Toxicol. 28 (2004) 181–186.

Samples were diluted to 10–100 times when the concentration was higher than 1000 ng/mL (nd, not detected).

4. Conclusion A speci?c method for the detection and quantitation of KT and its metabolite NK in urine by SPE and PCI–GC–MS was developed. According to the method validation data, the analytical method is suitable for the detection and quanti?cation of KT in human urine. Through the urinalysis of KT and its metabolites, the pattern of metabolism in human was distinguished from rat. Especially DHNK


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