Use of SPE and LC/TIS/MS/MS for rapid detection and quantitation of ketamine and its metabolite, norketamine, in urine

doi:10.1016/j.forsciint.2004.03.031

Forensic Science International

Volume 147, Issue 1, 6 January 2005, Pages 81-88

https://doi.org/10.1016/j.forsciint.2004.03.031 Get rights and content

Abstract

Ketamine (K) has become more and more popular for drug abuse in recent years. A lot of pre-treatment work such as extraction and derivatizing increase difficulties in the tests for ketamine in biological specimens. A rapid method to detect and quantitate ketamine and its metabolite norketamine in urine used deuterated dilution followed by solid phase extraction and liquid chromatography/TurboIonSpray/tandem mass spectrometry (LC/TIS/MS/MS) is described. Control recovery for both low and high concentrations can reach to 90%. Ten ketamine positive urines were examinated by this method. Concentrations ranged from 114 to 2925 ng/mL and from 453 to 9805 ng/mL for norketamine. The method was sensitive, specific, accurate and provided easy operation to detect and quantitate ketamine and its metabolites in urine.

Introduction

Ketamine is a dissociative medical anesthetic agent used in veterinary, human clinic [1], [2], and pediatric practices [3]. Both ketamine (K) and phencyclidine (PCP) have the same arylcyclo-alkylamine skeleton which produces similar hallucinogenic effects and is widely abused in “rave waves” of all night dance parties in the West [4]. There have been few abuse reports about these drugs in Taiwan until we first found in August of 2000, that four capsules of ketamine had been added to heroin powder. In the past 2 years, almost 10,000 g of ketamine powder and liquid (including attempted synthesis for illicit use, shipload smuggling of ketamine for drug abuse, mixing with another drug or alone in preparation for abuse in “rave party” cases) have been identified in our laboratory. The increasing drug abuse cases have received the government’s attention who placed ketamine into the controlled/scheduled III category in January 2002.

A biotransformation mechanism of ketamine was postulated by Chang and Glazko [5] (Fig. 1) in men and animals. Phase I reaction includes ketamine oxidation process (heteroatom demethylation) which yields norketamine and is followed by a hydroxylation process. It yields hydroxy-nor-ketamine (HNK) and results in only a small increase in hydrophilicity [6]. Phase II biotransformation reaction, which includes glucuronidation conjugation with glutathione and amino acid, results in a large increase in metabolite hydrophilicity that is easily excreted. Dehydronorketamine (DNK), which can be largely found in urine, is less hydrophilic than hydroxy-nor-ketamine and is considered a methodological artifact. There was no research about how much more DNK was found than norketamine in human hydro-bioliquids [7] until Moore et al. [8] detected a DNK presumptive concentration by LC/MS/MS (ion trap) in human urine. Whether DNK is a true metabolite of ketamine or an artifact is still being argued.

Confirmation tests of ketamine took place by GC/NPD [9],GC/MS and GC/CIMS [10]. The sample was derived with heptafluorobutyric anhydride (HFBA) and then was injected into GC/MS [11], HS-SPMEGC/MS [12], LC/UV [13], [14] and LC/MS single mass, tandem mass (ion trap) [8] in the past. There was no initial immunoassay test kit for ketamine. Therefore, a confirmation test of these drugs in forensic cases should be carried out with specific and accurate methods. Pre-treatment works in these tests also and should be perfomed carefully to prevent contamination and loss of the specimen. In ketamine’s metabolite analysis, the available DNK is expensive. There is less use for DNK as an indicator in routine detection. Hence ketamine-d4 and norketamine-d4 were chosen as internal standards using modified SPE from phencyclidine cleanup procedure and using sensitive and specific LC tandem mass (triple quadrupole) for analysis. Limit of quantitation (LOQ) was defined as the lowest concentration of calibration standards found in human urine with a precision of ±20% [15].

Consequently, we have found fewer and milder pre-treatment steps for urine, sensitive specific LC/MS/MS to help us to detect and quantify ketamines, and a basis for settling the argument of whether the compound is a true metabolite or an artifact.

Section snippets

Material

J.T. Baker LC grade solvents were used as mobile phase. All the reagents used were from Merck. D.I. water was prepared from distillation and filtration. All standards and internal standards were from Cerilliant including ketamine 1.0 mg/mL, ketamine-d4 100 μg/mL, Norketamine 1.0 mg/mL, and norketamine-d4 100 μg/mL.

Working standards were prepared from 1.0 mg/mL ketamine and norketamine standards (in methanol) including concentrations of 100 μg/mL, 10 μg/mL, 1 μg/mL, 100 ng/mL, 10 ng/mL, and 1 ng/mL.

Results and discussion

Multiple reaction monitoring (MRM) transition monitored for ketamine: 238m/z → 179m/z (quantitation), 238m/z → 220m/z (qualifying), 238m/z → 207m/z (qualifying), 238m/z → 189m/z (qualifying) 238m/z → 125m/z (qualifying), for norketamine: 224m/z → 207m/z (quantitation), 224m/z → 189m/z (qualifying), 238m/z → 179m/z (qualifying), 238m/z → 125m/z (qualifying). The ketamine’s product ion scan spectrum (PIS, Fig. 2a) of monitoring 238 (M + H)⁺ shows an initial loss of H₂O to give a fragment at 220,

Conclusion

A specific, sensitive and accurate method for detection and quantitation of ketamine and its metabolite norketamine by liquid chromatography tandem mass in human urine has been developed. The limit of detection quantitation for both ketamine and norketamine were 1.0 ng/mL with a urine sample volume of 1.0 mL. Four pairs of ions were chosen for confirmation by MRM; the prominent one was used for quantitation and the others were used for qualification. Analytes have good separation, and stability

Acknowledgements

The authors would like to thank Min-Chun Ho for helpful comments and discussion. We would like to acknowledge our past Director, Wun Jing-Hui, for his support during this study.

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Use of SPE and LC/TIS/MS/MS for rapid detection and quantitation of ketamine and its metabolite, norketamine, in urine

Abstract

Introduction

Section snippets

Material

Results and discussion

Conclusion

Acknowledgements

Brit. J. Anesth

J. Chromatogr

J. Chromatogr. B

J. Am. Soc. Mass Spectrom

J. Biol. Chem

Ketamine—its pharmacology and therapeutic uses

Anesthesiology

Gsilvay, ketamine: an update on the first twenty five years of clinical experience

Can. J. Anaesth

Emergency department use of ketamine in pediatric status asmaticus

J. Asthma

Club drug: methylenedioxymethamphetamine, flunitrazepam, ketamine hydrochloride, and gamma-hydroxybutyrate

Am. J. Health Syst. Pharm