Quantitative detection of ketamine, norketamine, and dehydronorketamine in urine using chemical derivatization followed by gas chromatography–mass spectrometry

Abstract

A repeatable and highly sensitive analytical method using gas chromatography–mass spectrometry (GC–MS) in the selected ion monitoring mode (SIM) is developed for the simultaneous detection of ketamine (KT), norketamine (NK), and newly introduced dehydronorketamine (DHNK) in urine. The test specimen along with the deuterium analogues as internal standards (IS): d₄-KT for KT and d₄-NK for NK/DHNK, was extracted on an automatic solid-phase extraction (SPE) apparatus. The extracted eluate then was dried and derivatized with N-methyl-bis(trifluoroacetamide) (CF₃CONCH₃COCF₃, MBTFA). Finally, the cooled derivatized solution was directly injected into the GC–MS system for analysis. The proposed process achieves high sensitivity for the detection of KT, NK, and DHNK. Correlation coefficients derived from typical calibration curves in the range of 20–2000 ng/mL are 1.000 for KT and NK, 0.999 for DHNK. The limits of detection (LODs) and limits of quantitation (LOQs) are 0.5–1.0 and 1.5–3.0, respectively. The overall method recoveries of KT, NK, and DHNK are 82.2–93.4. The intra- and inter-day run deviations are smaller than 5.0%. The analytical scheme was also applied to the determination of KT, NK, and DHNK in 20 KT suspected urine specimens, and the results reconfirm that DHNK is a main metabolite of KT.

Introduction

Ketamine (KT) is catalogued as a synthetic anesthetic for human and veterinary surgery [1], it induces sedation, immobility, and amnesia. The drug is often called a dissociative anesthetic because a trancelike and cataleptic state with amnesia occurs with adequate doses [2], [3]. KT is also structurally and pharmacologically related to phencyclidine [4], and is capable of producing some hallucination effects similar to those produced by phencyclidine. It was initially abused by medical personnel for its hallucinogenic effects, and gradually became popular on the European party scene in the early 1990s, then spread to other parts of the world. KT was stated to be metabolized to at least two major compounds of pharmacological interest: to norketamine (NK) by N-demethylation, which then is converted to dehydronorketamine (DHNK) by dehydrogenation [5], although some articles suggest that DHNK is an artifact resulting from the gas chromatography (GC) or gas chromatography–mass spectrometry (GC–MS) temperature programming process [6], [7]. Analysis of KT and/or its demethylated metabolites has been accomplished by high-performance liquid chromatography [8], [9], [10], [11], [12] and liquid chromatography–mass spectrometry methods [13]. The analytes were also detected by gas chromatography with flame ionization detection [14], [15], nitrogen phosphate detection [16], electron-capture detection [5], or by mass spectrometry detection with derivatization [6] or without derivatization [17], [18], [19], [20], [21], [22]. Most of the above methods involved liquid–liquid extraction (LLE) or solid-phase extraction (SPE) for sample preparation; a method combining solid-phase microextraction and GC–MS has been reported, too [23]. KT is a seriously stereo-hindered secondary amine and is difficult to derivatize by most derivatizing reagents, so only one method using chemical derivatization (ChD) for KT urine testing was reported recently [6]. That article described the quantitative analysis of KT and NK using GC–MS preceded by LLE and pentafluorobenzoyl chloride (PFBC) derivatization. The current study explores a new and simplified approach in which the extraction is operated by automatic SPE equipment and the analytes are derivatized effectively by N-methyl-bis(trifluoroacetamide) (CF₃CONCH₃COCF₃, MBTFA). There have been some articles reporting that DHNK is an artifact of KT in the GC or GC–MS process [6], [7]. Although some report said that DHNK is a metabolite of KT, yet it also suggested that DHNK may be an analytical artifact rather than a metabolite [24]. Because DHNK was not commercially available, almost none of the articles studied the quantitation of DHNK using GC–MS methods, and it is uncertain whether DHNK is a metabolite or an artifact. In this study, the protocol developed to simultaneously analyze KT, NK, and DHNK generates excellent recovery and assay linearity, and is applied to reconfirm that DHNK is an actual metabolite of KT when they are analyzed using GC–MS.