Quantitative chiral and achiral determination of ketamine and its metabolites by LC–MS/MS in human serum, urine and fecal samples
Introduction
Ketamine (KET) is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist which is frequently used as intravenous dissociative anesthetic agent that has also been shown to induce analgesia by non-opioid mechanisms [1], [2].
KET is metabolized to norketamine (n-KET) (main active metabolite), 5,6-dehydronorketamine (DHNK) and various hydroxymetabolites by cytochrome P450 enzymes [3], [4]. KET is a chiral compound that is usually available as a racemate. However, the S (+)-KET was shown to be more potent than the R(−)-enantiomer and exhibits a higher clearance and faster anesthetic recovery compared to S (+)-KET [5], [6]. It was reported that both enantiomers have different pharmacokinetic profiles [7].
In addition to its frequent use as anesthetic, ketamine becomes of interest for the treatment of other indications such as pain or depression [8], [9], [10], [11]. In this regard, 2R,6R-hydroxynorketamine (2R,6R-HNK) was recently shown to be responsible for the antidepressant activity of KET most likely via activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [12], while the other isomer 2S,6S-hydroxynorketamine (2S,6S-HNK) acts as a selective α7-nicotinic acetylcholine (α7-nACh) receptor inhibitor [13]. Consequently, both compounds may contribute substantially to the KET pharmacodynamics and should be monitored in analytical assays for KET in humans.
Established quantitative assays for KET and n-KET racemate or enantiomers in human and animal plasma were done using HPLC with UV-detection [14], [15], [16], [17], [18], [19]. Furthermore, gas chromatography [20], [21], [22] or gas chromatography-mass spectrometry methods [23], [24] have also been reported. As an alternative to these methods, enantioselective capillary electrophoresis assays for analysis of KET and n-KET enantiomers were developed [25], [26], [27], [28]. However, the limitation of the aforementioned methods was their limited sensitivity with LLOQ ranging from 10 to 40 ng/ml and partly their lack of specificity.
To overcome these limitations, more sensitive and specific LC–MS/MS methods for KET and n-KET have been developed [29], [30], [31], [32]. Thereby, the sensitivity could be markedly increased (LLOQ up to 1–50 ng/ml) which enabled monitoring of serum concentration-time curves of KET and n-KET after administration of lower IV doses of KET as used in the treatment of pain or depression [29], [30], [31], [33], [34]. However, as considerable limitations, these assays required up to 500 ml plasma, used time-consuming and costly sample preparation methods (mostly solid phase extraction) and the chromatographic run time was up to 30 min (Table 1).
Some of the aforementioned assays enable the stereoselective quantification of KET and n-KET [30,31]while only one of these previously published methods also considered other relevant KET metabolites including DHNK and HNK [30]. As stated above, especially 2R,6R-HNK and 2S,6S-HNK are of interest today according to their antidepressant activity. However, so far, the 12 HNK isomers differing in constitution and/or stereochemistry were determined only in an achiral manner, leaving the separation of the 6 pairs of enantiomers unresolved; i.e. 2S,6S;2R,6R-HNK and 2S,6R;2R,6S-HN [30], [33], [34].
Thus, it was the aim of our study to develop and validate LC–MS/MS assays for the achiral and stereoselective quantification of KET, n-KET, DHNK and HNK in serum, urine and feces enabling the characterization of the KET pharmacokinetics after administration of a low intravenous dose (5 mg vs. the standard dose of 70–140 mg). In order to fulfill these criteria, we developed and validated three LC–MS/MS assays; one for the quantification of racemic KET, n-KET and DHNK in human serum, urine and feces and two additional methods for the separation and quantification of the S- and R-enantiomers of KET, n-KET, DHNK and HNK. With respect to the latter, we succeeded for the first time to determine in a enantioselective manner the antidepressant active KET metabolites 2S,6S-hydroxynorketamine (2S,6S-HNK) and 2R,6R-hydroxynorketamine (2R,6R-HNK) with the ability to separate and detect 10 additional hydroxylated norketamine metabolites.
Section snippets
Chemicals
Isopropanol was obtained in LC–MS quality from Carl Roth (Karlsruhe, Germany). Acetonitrile was bought in LC–MS quality (Chromasolv®, Sigma–Aldrich, Taufkirchen, Germany). Deionized water (conductance: ≤0.055 μS/cm, pH 5.0–6.0) was generated with the Astacus® system (membrapure, Hennigsdorf, Germany). The internal standards D4-Ketamine and D4-norketamine as well as KET, n-KET and DHNK were from Sigma–Aldrich (Taufkirchen, Germany) and 2R,6R-HNK and 2S,6S-HNK were kindly provided by the National
Results and discussion
In this report, we describe the development and validation of three LC–MS/MS assays for the quantification of KET and its major metabolites n-KET, DHNK and HNK in human serum, urine and feces which enable racemic and enantioselective determination of the analytes. Compared to previously published methods, our assays possess some superior features such as a lower volume of required biological matrix, an increased analytical sensitivity and a shortened chromatographic run time (Table 1).
In our
Application of the methods
Our method was successfully applied to quantify KET, n-KET, DHNK and the respective R- and S-enantiomers in serum, urine and feces as well as two relevant HNK enantiomers in samples from a pharmacokinetic pilot study performed in one healthy volunteer who received 5 mg KET for 30 min as intravenous infusion. To our knowledge, this is the first time to show separation of the active metabolites 2R,6R-HNK and 2S,6S-HNK from other HNK isomers in human serum and urine and to apply this method in a
Conclusion
We successfully developed, validated and applied three analytical assays to quantify KET, and its metabolites in a chiral and achiral manner in human serum, urine and feces. Our method could slightly improve the analytical features of previously published methods (e.g. higher sensitivity) and enabled for the first time the separation and quantification of the two enantiomers 2R,6R-HNK and 2S,6S-HNK in serum and urine which are responsible for the antidepressant activity of KET. The methods were
Conflict of interest
The authors declare that they have no conflict of interest.
Financial support
The analytic part was supported by the grant 03IPT612X (InnoProfile) of the German Federal Ministry for Education and Research.
The clinical study has been sponsored by an institutional grant of Develco Pharma Schweiz AG, Pratteln, Switzerland.
Mahmoud Hasan was sponsored by Future University in Egypt (Hasan Azazy scholarship).
Acknowledgements
The authors thank Sabine Bade, Gitta Schumacher and Danilo Wegner for excellent technical assistance. The analytical standards for the identification and quantification of the metabolites 2R,6R-hydroxynorketamine (NCGC00378227) and 2S,6S-hydroxynorketamine (NCGC00373033) were kindly provided by the National Center for Advancing Translational Sciences (NCATS).
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Department of Pharmaceutical Chemistry Future University in Egypt, End of 90th road, eltagamoa el-Kahmes, New Cairo, Cairo, Egypt.