Chiral Separation and Determination of Enantiomer Elution Order of Novel Ketamine Derivatives Using CE-UV and HPLC-UV-ORD

Abstract

Besides the well-known hallucinogenic ketamine, various novel ketamine derivatives are available on the illicit drug market, sold as designer drugs. Minor chemical changes to the parent compound aim to circumvent existing narcotic drug laws while mimicking the effects of the original substance. Ketamine and some of its derivatives possess a chiral centre and therefore exist as two enantiomers. While differences in the effects of S- and R-ketamine are well studied, this is not the case for ketamine derivatives. Therefore, the development and adaptation of suitable enantioseparation methods for those compounds is important to face the problems of the constantly changing drug market. In this study, different chiral separation methods for capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were tested on 11 ketamine derivatives. Some of them were enantioseparated for the first time due to their novelty. All compounds were at least partially separated on both instruments. HPLC separations were conducted using four different polysaccharide-based chiral stationary phases. Furthermore, an optical rotation detector coupled to the HPLC enabled the determination of the enantiomer elution order. In CE analysis, enantioseparation was achieved using 2% (w/v) acetyl-β-cyclodextrin or carboxymethyl- β-cyclodextrin in 10 mM di-sodium hydrogen phosphate as the background electrolyte in capillary electrophoresis.

1. Introduction

After the first clinical trials in 1966 [1], ketamine was officially authorized for medical use a few years later in 1970 by the Food and Drug Administration (FDA) [2]. Shortly afterwards, in the middle of the 1970s, it appeared on the illegal drug market and has remained popular since then. Besides the analgesic and anesthetic effects, the psychedelic potential of ketamine provoked interest for recreational use [3]. Nevertheless, ketamine is deemed to be an essential medicine by the World Health Organization (WHO) [4], mainly used as an analgesic and anesthetic in human and veterinary medicine [3,5]. Several studies in recent years have also shown its suitability for the acute treatment of otherwise therapy-resistant depression, [6] which led to the authorization of an esketamine containing nose-spray as a treatment in psychiatric emergencies [7]. Esketamine is the eutomer of ketamine; it is the S-(+)-enantiomer and about three to four times more potent than the R-(-)-enantiomer, which allows the same anesthetic effect at lower dosages in comparison to the racemic mixture, while, at the same time, unwanted side effects are reduced [8,9].

Determining the enantiomeric status of a ketamine-containing sample, either for police or for consumer drug checking services, helps to assume the origin of the sample. The pure S-(+)-enantiomer may indicate the theft or illegal acquisition of esketamine for intended medical purposes, whereas samples containing the racemic mixture mostly originate from illegal ketamine laboratories, though diversion from medical resources is also a possible source [10,11]. Furthermore, enantiomer analysis for consumer drug checking can help to reduce overdosing. A chiral HPLC method is also part of the purity test for esketamine in the European Pharmacopoeia [12]. Additionally, the determination of specific optical rotation is used for the identification of racemic ketamine [13], as well as esketamine [12].

The European Union Drugs Agency (EUDA) has seen an increasing availability of ketamine on the drug market and a related increase in health problems within recent years [10,11]. Recently, an intensively pink powder, sold as “Pink Cocaine” (Figure 1), turned out to represent a mixture of ketamine with MDMA (3,4-methylenedioxymethamphetamine, “Ecstasy”) or other stimulants like amphetamine, rather than cocaine [11]. This unpredictable mix of substances and the deceptive declaration may also mislead consumers toward unintended ketamine consumption and the miscalculation of dosages.

For a couple of years, other new ketamine derivatives have been available on the illegal drug market. They are created to mimic the effects of their model compound while simplifying availability. Due to chemical modifications like adding or removing substituents, a new structure is created that is not listed within national narcotic drug laws. By the end of 2023, 27 different arylcyclohexylamines (ketamine-derivatives) had been registered by the EUDA [11].

Some of them are chiral structures, and the differences in effect from S-ketamine and R-ketamine may also similarly apply for these derivatives. Therefore, implementing enantioseparation techniques, not only for ketamine but also for its recreationally used derivatives is of great interest. There are already different approaches available for the chiral separation of S-ketamine and R-ketamine using cyclodextrins in capillary electrophoresis (CE) [14,15,16,17,18,19,20,21], but so far, to the best of our knowledge, only limited data on the enantioseparation of ketamine derivatives are available [22].

Among the different chiral selectors suitable for CE, cyclodextrins are commonly used and have proven their applicability in the enantioseparation of different substance classes [23]. Cyclodextrins are cyclic oligosaccharides built up from six (α-cyclodextrins), seven (β-cyclodextrins), or eight (γ-cyclodextrins) glucopyranose units. In CE, they can be used as additives to the background electrolyte (BGE), as many of the various available cyclodextrin derivatives are highly soluble in water [23,24]. The enantioseparation of ketamine and four derivatives using cyclodextrin-assisted CE was conducted by Hägele et al. in 2020 [22].

In the enantioseparation of NPS including ketamine derivatives with high-performance liquid chromatography (HPLC), polysaccharide-based chiral stationary phases (CSPs) play an important role. In recent years, different chiral selectors have been tested. In 2018, ketamine and three derivatives were successfully enantioseparated using a Lux^® i-cellulose-5 column [25]. In 2019, five ketamine derivatives were separated using a Lux^® i-Amylose-1 column under normal phase conditions [26]. With a Lux^® AMP column, ketamine and five derivatives were tested, and, except from methoxetamine, all of them were enantioseparated [27]. For ketamine itself and its metabolites, a variety of methods have been developed over the years, using polysaccharide-based CSPs, as well as other CSPs [28,29,30,31,32,33,34,35,36,37,38]. In 2009, experiments with sulfated β-cyclodextrin as a mobile phase additive for the separation of ketamine and other chiral pharmaceuticals were conducted [16].

To the best of our knowledge, only limited data on the enantioseparation of recently emerged ketamine derivatives are available. For this reason, the present publication aims to supplement existing analytical approaches with methods for the latest derivatives. Eleven substances from the class of ketamines were examined. The main focus in this paper is to test different chiral selectors in HPLC and CE, both with UV detection for the enantioseparation of ketamine derivatives. Additionally, an optical rotation detector (ORD) coupled to the HPLC is used to show the rotation descriptors (+) or (−) of the analyzed ketamines, and though it cannot reveal the absolute configuration (S) or (R), it helps to determine their enantiomer elution order (EEO).

2. Materials and Methods

Eleven different ketamine derivatives and a solution of enantiopure S-(+)-ketamine, all present as hydrochloric salts, were measured with CE as well as HPLC. Ketamine was bought from Fagron GmbH (Glinde, Germany) and S-(+)-ketamine was bought as Ketanest^® S 5 mg/mL ampoules (Pfizer Manufacturing, Puurs, Belgium) acquired via Kwizda Pharma Distribution (Vienna, Austria). The investigated ketamine derivatives were obtained from various online traders, selling NPS for recreational use, or were real-life samples provided by the Austrian Police for research purposes. The identity of all compounds was checked with gas chromatography–mass spectrometry (GC-MS) and, if necessary, with nuclear magnetic resonance (NMR) spectroscopy prior to measurements. The names and structures of all tested compounds are displayed in Table 1.

2.1. Capillary Electrophoresis

2.1.1. Sample Preparation

About 1 mg of each sample was dissolved in 1 mL of water. The content from a Ketanest^® S 5 mg/mL ampoule, which contained S(+)-ketamine dissolved in a physiological saline solution, was diluted to 1 mg/mL with analytical-grade water (Fisher Scientific, Loughborough, UK).

2.1.2. Instrumentation and Method

Measurements were carried out on an Agilent 7100 Capillary Electrophoresis System (Agilent, Santa Clara, CA, USA) using single-wavelength UV detection at 210 nm. The voltage to cathode was set to 25 kV, and the cassette temperature was 25 °C. The measurements were performed in a fused silica capillary (ID 50 µm) purchased from MicroQuartz (Munich, Germany) with a total length of 68.5 cm and an effective length of 60 cm. Sample injection was conducted dynamically by applying 10 mbar pressure for 5 s on the inlet vial.

Experiments prior to this work based on Hägele et al. [22] determined acetyl-β-cyclodextrin and carboxymethyl-β-cyclodextrin to be suitable chiral selectors for ketamine derivatives. Acetyl-β-cyclodextrin (degree of substitution = 1.0) was acquired from Wacker-Chemie GmbH (Salzburg, Austria); carboxymethyl-β-cyclodextrin (degree of substitution = 3.5) was from CycloLab Ltd. (Budapest, Hungary).

The background electrolyte consisted of aqueous 10 mM di-sodium hydrogen phosphate (Merck KGaA, Darmstadt, Germany) and 2% (w/v) of each cyclodextrin, respectively. To help in dissolving the cyclodextrins, an ultrasonic bath was used for a few minutes. The pH was adjusted to 2.5 with diluted phosphoric acid (Merck KGaA, Darmstadt, Germany). The BGE solutions were filtered through a 0.45 µm single-use syringe filter (Carl Roth, Karlsruhe, Germany) to remove remaining suspended particles.

2.2. High-Performance Liquid Chromatography

2.2.1. Chemicals and Reagents

All chemicals were of analytical reagent grade. Isopropanol (2-PrOH) and n-hexane were purchased from Fisher Scientific (Loughborough, UK). Ethanol (EtOH) was from Merck KGaA (Darmstadt, Germany). Methanol and diethylamine (DEA) were obtained from VWR (Fontenay-sous-Bois, France).

2.2.2. Chromatographic Conditions

Measurements were performed on an Agilent 1260 Infinity II liquid chromatograph (Agilent, Santa Clara, CA, USA) equipped with a variable wavelength detector and a Jasco OR-4090 optical rotation detector (Jasco Co., Tokyo, Japan). UV data were collected at 254 nm. Measurements were performed under isocratic conditions at 25 ± 1 °C; the injection volume was 1 µL, and the flow rate was 1 mL/min. Two different mobile phase compositions were used, namely, n-hexane–2-PrOH–DEA and n-hexane–EtOH–DEA, both in a ratio of 95:5:0.1 (v/v/v). Four different polysaccharide-based chiral stationary phases (CSPs) by Phenomenex (Torrance, CA, USA) were used, and they are described in Table 2.

2.2.3. Sample Preparation

Approximately 1 mg of samples, all present as hydrochloric salts, were dissolved in 1 mL of methanol. For a few measurements, some samples were dissolved in 2-PrOH. For some samples, the use of an ultrasonic bath was necessary to reach full dissolution. The solution of S-(+)-ketamine was prepared by diluting the solution of 5 mg/mL with MeOH to reach a final concentration of 1 mg/mL.

3. Results and Discussion

3.1. CE Experiments

All ketamine derivatives were either baseline or partially separated using acetyl-β-cyclodextrin (2%, w/v) as a chiral selector. All samples were detected within 17 min. Additionally, spiking ketamine with the S-(+)-enantiomer determined the enantiomeric migration order of R-ketamine before S-ketamine. Using carboxymethyl-β-cyclodextrin (2%, w/v) as the chiral selector, only seven partial separations and one baseline separation were obtained within 19 min. However, the resolutions of N-ethylketamine and 2F-2-oxo-PCE were doubled compared to using acetyl-β-cyclodextrin as the selector. Both substances possess an N-ethyl group and a halogenated benzene ring. Besides ketamine itself, the lowest-resolution values (0.63 and 0.47) using acetyl-β-cyclodextrin were achieved for them. A comparison of both the electropherograms of N-ethylketamine using (A) carboxymethyl- or (B) acetyl-β-cyclodextrin as an additive in the buffer is shown below, in Figure 2.

Both cyclodextrins were investigated under the voltage conditions of +25 kV and +27 kV. An applied voltage of +25 kV slightly extended the time of measurements for 2 to 3 min but improved the resolution of the enantiomers; hence, it was chosen for the final optimized method. The best result under the final conditions was obtained for hydroxetamine, with a resolution value of 5.41 using acetyl-β-cyclodextrin as the chiral selector. The enantioseparation of hydroxetamine is shown in Figure 3.

The separation results including migration times, apparent separation factors α_app (t₂/t₁), and resolution values—calculated as Rs = 1.177 × ((t₂ − t₁)/(W_0.5h1 + W_0.5h2))—for the investigated samples, comparing both cyclodextrins used with the voltage set to +25 kV, are shown in Table 3a,b.

3.2. HPLC Experiments

3.2.1. Comparison of Separation Ability of Different CSPs and Mobile Phase Compositions

Table 4a,b show the separation results given as retention times, separation factor (k₂/k₁) and chromatographic resolution—calculated as Rs = 1.177 × ((t₂ − t₁)/(W_0.5h1 + W_0.5h2))—for the tested CSPs under the mobile phase compositions n-hexane–2-PrOH–DEA 95:5:0.1 (v/v/v) and n-hexane–EtOH–DEA 95:5:0.1 (v/v/v), respectively. The CSP with the highest Rs value for each mobile phase condition is highlighted in bold letters.

Figure 4 gives a summary of HPLC results under all tested conditions, where numbers of baseline-separated, partially separated and non-separated substances are given.

AMY-3 showed the best results under both mobile phase conditions. All tested compounds were at least partially separated. For 2-PrOH, Rs values ranged between 0.94 and 7.22; the EtOH range was between 1.16 and 27.65. The highest value was achieved for deschloroketamine, with 18 min between the two peaks. Generally, the two amylose CSPs showed better separation ability for the set of compounds compared to cellulose columns. The reduced separation ability of AMY-1 compared to AMY-3 may be partly due to the larger particle size of 5 μm. CEL-5 produced the highest number of non-separated compounds. For 2-PrOH mobile phase, 9 out of 11 substances showed the highest resolution with AMY-3; for EtOH, it was 8 out of 11.

The results were also investigated regarding mobile phase composition. Considering measurement results with all four CSPs, 8 out of 44 measurements with EtOH and 3 out of 44 with 2-PrOH led to no separation. In total, 20 and 21 baseline separations were achieved with EtOH and 2-PrOH, respectively. Even though no general advantage of one of the two mobile phase compositions was found, there were some very strong differences in the individual results for the 11 substances. For example, hydroxetamine was baseline-separated with an Rs value of 4.58 using 2-PrOH and CEL-5 but showed only partial separation (Rs = 0.73) with EtOH and CEL-5. For 2F-ketamine, the exchange of 2-PrOH with EtOH led to an increase in resolution from 1.35 to 5.37 with AMY-3. These examples show that the two mobile phase compositions can be used complementarily. In combination with the different CSPs, all tested compounds were baseline-separated under at least one condition.

3.2.2. Influence of Chiral Selector and Mobile Phase Composition on Enantiomer Elution Order

Figure 5 shows the enantiomer elution order of the 11 tested compounds with four CSPs and two mobile phases each. The enantiomer ((+) or (−)) that eluted first is indicated.

In the OR chromatograms, a positive signal for the (+)-enantiomers and a negative signal for the (−)-enantiomers was received. Figure 6 shows the comparison of pure S-(+)-ketamine and racemic ketamine. For S-ketamine, only the positive signal is visible.

For the chiral columns AMY-3 and CEL-4, both containing two different substituent groups on the phenylcarbamate residue, the replacement of 2-PrOH with EtOH in the mobile phase showed no impact on the EEO. In the case of AMY-1 and CEL-5, containing only methyl- or chloride groups, respectively, changes in the EEO were observed for some substances when changing the mobile phase composition. This effect was especially observed with substances that showed a low resolution. Regarding results for all CSPs, in 32 out of 44 cases, no EEO reversal was observed when exchanging 2-PrOH with EtOH.

The influence of mobile phase composition on EEO had already been determined in different studies with polysaccharide-based CSPs for other substance classes [39,40,41,42,43,44]. In these studies, it was shown that not only the type but also the percentage of cosolvent in the mobile phase had an impact on the EEO. It was found that the polarity of the mobile phase was a crucial factor in determining the EEO [39].

With both cellulose columns, (−)-enantiomers eluted first for more than 50% of all measurements, whereas for amylose columns, a tendency towards (+)-enantiomers as first eluents was observed. In many cases, the EEO changed when switching from amylose to cellulose under the same mobile phase conditions. Figure 7 and Figure 8 show the chromatograms of deschloroketamine with column AMY-1 compared to CEL-5, where the EEO is reversed.

Concerning relations between structural properties and EEO, the following conclusions could be drawn. Ketamine and N-ethylketamine, differing only in the length of the N-alkyl residue, showed the same EEO under all tested conditions. The same was true for methoxetamine and methoxpropamine. Deschloroketamine and 2F-ketamine, which differ only in the presence or absence of a fluorine atom on the phenyl ring, also showed the same behaviour. For 2-Oxo-PCE and 2F-2-Oxo-PCE, this was also observed.

For compounds not visually separated in the UV chromatograms, the EEO could still be determined in OR chromatograms, as positive and negative peak signals for enantiomers were still visible. For example, Figure 9 shows the UV and OR chromatogram of methoxetamine with CEL-5 and EtOH as the mobile phase component. The OR chromatogram shows that the (+)-enantiomer eluted slightly earlier. Therefore, the OR detector can be a useful tool to check the enantiomeric purity, when peaks apparently coelute, as the two signals overlap in the UV chromatogram. To verify this, measurements of the two substances ketamine and methoxetamine with an achiral RP-8 column (LiChrospher^® 100 RP-8 endcapped, 125-4 (5 µm); Merck KGaA, Darmstadt, Germany) were performed. In both cases, no analyte signals were received in the OR chromatogram, as no chiral recognition took place.

4. Conclusions

Using capillary electrophoresis for chiral separations is an inexpensive and quick method to determine possible chiral selectors for the substances of interest, especially for the variety of cyclodextrins available, before investing in chiral columns for HPLC. Furthermore, it represents a green approach, because of the low solvent amounts needed.

The determination of the EEO can be a very important tool when creating a new method for the quantification of enantiomer impurities. With HPLC coupled to the ORD, it is possible to determine the EEO in a racemic mixture when no enantiopure standard is available. With different CSPs and mobile phase conditions, a suitable method for all tested ketamine derivatives was found and can also be expanded to cover new derivatives in the future.

Enantioseparation experiments on a small scale, using either CE or analytical HPLC, can be upscaled for preparative chiral separations. Pure enantiomers can further be used for pharmacological studies to determine their psychoactive properties.

Author Contributions

Conceptualization, E.-M.H. and E.S.; methodology, E.-M.H. and E.S.; investigation, E.-M.H. and E.S.; resources, M.G.S.; writing—original draft preparation, E.-M.H. and E.S.; writing—review and editing, E.-M.H., E.S. and M.G.S.; visualization, E.-M.H. and E.S.; supervision, M.G.S.; project administration, M.G.S. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

The authors acknowledge financial support from the University of Graz.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1. Ketamine-MDMA mixture sold as Pink Cocaine seized by Austrian Police.

Figure 2. Enantioseparation of N-ethylketamine using (A) carboxymethyl- or (B) acetyl-β-cyclodextrin as chiral selector with voltage set to +25 kV.

Figure 3. Enantioseparation of hydroxetamine using 2% (v/w) acetyl-β-cylcodextrin in an aqueous 10 mM di-sodium hydrogen phosphate background electrolyte, with voltage set to +25 kV.

Figure 4. Summary of separation results under all tested conditions.

Figure 5. EEO with elution of first enantiomer indicated.

Figure 6. (A) UV and (B) OR chromatogram of racemic ketamine, (C) UV and (D) OR chromatogram of S-(+)-ketamine with the CSP AMY-3 and a mobile phase of n-hexane:2-PrOH:DEA (95:5:0.1); (−) and (+) indicate substance peaks.

Figure 7. (A) UV and (B) OR chromatogram of deschloroketamine with the CSP AMY-1 and a mobile phase of n-hexane–2-PrOH–DEADEA (95:5:0.1); (−) and (+) indicate substance peaks.

Figure 8. (A) UV and (B) OR chromatogram of deschloroketamine with the CSP CEL-5 and a mobile phase of n-hexane–2-PrOH–DEA (95:5:0.1); (−) and (+) indicate substance peaks.

Figure 9. (A) UV and (B) OR chromatogram of methoxetamine with the CSP CEL-5 and a mobile phase of n-hexane–EtOH–DEA (95:5:0.1); (−) and (+) indicate substance peaks.

Table 1

Names and structures of all tested compounds.

Ketamine2-(2-Chlorophenyl)-2-(methylamino)cyclohexan-1-one[Image omitted. Please see PDF.]	Deschloroketamine2-Phenyl-2-(methylamino)cyclohexan-1-one[Image omitted. Please see PDF.]	2F-Ketamine2-(2-Fluorophenyl)-2-(methylamino)cyclohexan-1-one[Image omitted. Please see PDF.]	N-Ethylketamine2-(2-Chlorophenyl)-2-(ethylamino)cyclohexan-1-one[Image omitted. Please see PDF.]
2-Oxo-PCE2-(Ethylamino)-2-phenylcyclohexan-1-one[Image omitted. Please see PDF.]	Methoxetamine2-(Ethylamino)-2-(3-methoxyphenyl)cyclohexan-1-one[Image omitted. Please see PDF.]	Hydroxetamine(3-HO-2-Oxo-PCE)2-(Ethylamino)-2-(3-hydroxyphenyl)cyclohexan-1-one[Image omitted. Please see PDF.]	Methoxpropamine(MXPr)2-(3-Methoxyphenyl)-2-(propylamino)cyclohexan-1-one[Image omitted. Please see PDF.]
Methoxisopropamine (MXiPr)2-(Isopropylamino)-2-(3-methoxyphenyl)cyclohexan-1-one[Image omitted. Please see PDF.]	Deoxymethoxetamine(DMXE)2-(Ethylamino)-2-(3-methylphenyl)cyclohexan-1-one[Image omitted. Please see PDF.]	2F-2-Oxo-PCE2-(Ethylamino)-2-(2-fluorophenyl)cyclohexan-1-one[Image omitted. Please see PDF.]

Table 2

Parameters of CSPs used for experiments.

Abbreviation	Trade Name	Chiral Selector	Column Dimensions and Particle Size
AMY-1	Lux® i-Amylose-1	Amylose tris(3,5-dimethylphenylcarbamate	250 × 4.6 mm I.D.; 5 μm
AMY-3	Lux® i-Amylose-3	Amylose tris(3-chloro-5-methylphenylcarbamate	250 × 4.6 mm I.D.; 3 μm
CEL-5	Lux® i-Cellulose-5	Cellulose tris(3,5-dichlorophenylcarbamate	250 × 4.6 mm I.D.; 3 μm
CEL-4	Lux® Cellulose-4	Cellulose tris(4-chloro-3-methylphenylcarbamate	250 × 4.6 mm I.D.; 3 μm

Table 3

(a) Enantioseparation results for ketamine derivatives using 2% (w/v) acetyl-β-cyclodextrin as the chiral selector in CE. (b) Enantioseparation results for ketamine-derivatives using 2% (w/v) carboxymethyl-β-cyclodextrin as the chiral selector in CE.

(a)
Compound	t1 (min)	t2 (min)	αapp (t2/t1)	Rs
Ketamine	14.13	14.26	1.009	0.50
Deschloroketamine	12.53	13.21	1.054	2.70
2F-Ketamine	12.80	13.05	1.020	1.35
N-Ethylketamine	14.44	14.58	1.010	0.63
2-Oxo-PCE	13.94	14.68	1.053	3.17
Methoxetamine	14.19	15.20	1.072	3.34
Hydroxetamine (3-HO-2-oxo-PCE)	14.59	16.10	1.103	5.41
Methoxpropamine (MXPr)	13.78	14.27	1.035	1.96
Methoxisopropamine (MXiPr)	15.45	15.98	1.034	1.82
Deoxymethoxetamine (DMXE)	13.24	13.54	1.023	1.24
2F-2-Oxo-PCE	13.68	13.78	1.007	0.47
(b)
Compound	t1 (min)	t2 (min)	αapp (t2/t1)	Rs
Ketamine	16.77	-	-	-
Deschloroketamine	15.06	15.31	1.016	1.08
2F-Ketamine	15.41	15.46	1.003	0.21
N-Ethylketamine	18.04	18.38	1.019	1.28
2-Oxo-PCE	15.35	15.44	1.006	0.42
Methoxetamine	18.93	19.37	1.023	1.28
Hydroxetamine (3-HO-2-oxo-PCE)	18.32	18.93	1.033	2.10
Methoxpropamine (MXPr)	16.64	-	-	-
Methoxisopropamine (MXiPr)	19.65	19.74	1.005	0.34
Deoxymethoxetamine (DMXE)	18.31	-	-	-
2F-2-Oxo-PCE	16.34	16.59	1.015	1.07

Table 4

(a) Separation results for the tested CSPs using n-hexane:2-PrOH:DEA (95:5:0.1) as mobile phase. (b) Separation results for the tested CSPs using n-hexane:EtOH:DEA (95:5:0.1) as mobile phase.

(a)
Compound	t1 (min)	t2 (min)	α	Rs	CSP
Ketamine	8.31	8.62	1.06	1.11	AMY-1
	14.40	15.80	1.12	3.60	AMY-3
	21.67	21.94	1.01	0.48	CEL-5
	12.50	13.16	1.07	1.64	CEL-4
Deschloroketamine	8.04	10.24	1.44	6.96	AMY-1
	13.82	14.17	1.03	0.94	AMY-3
	15.01	16.87	1.16	3.91	CEL-5
	10.49	11.77	1.17	3.66	CEL-4
2F-Ketamine	8.57	9.47	1.16	3.00	AMY-1
	16.34	16.91	1.04	1.35	AMY-3
	22.08	23.50	1.08	2.12	CEL-5
	13.13	15.62	1.25	5.64	CEL-4
N-Ethylketamine	5.72	5.77	1.02	0.31	AMY-1
	8.72	9.56	1.15	3.49	AMY-3
	12.06	n.d.	-	-	CEL-5
	7.60	8.05	1.10	1.71	CEL-4
2-Oxo-PCE	5.19	5.45	1.12	1.46	AMY-1
	7.50	8.79	1.29	6.19	AMY-3
	7.90	n.d.	-	-	CEL-5
	6.30	6.59	1.09	1.50	CEL-4
Methoxetamine	6.41	6.59	1.05	0.87	AMY-1
	10.67	12.47	1.24	5.71	AMY-3
	10.07	10.50	1.06	1.38	CEL-5
	7.47	7.89	1.10	1.74	CEL-4
Hydroxetamine	11.17	13.69	1.31	4.95	AMY-1
	18.19	22.21	1.27	7.22	AMY-3
	11.95	14.09	1.24	4.58	CEL-5
	12.88	n.d.	-	-	CEL-4
Methoxpropamine	6.04	6.12	1.03	0.80	AMY-1
	8.73	10.48	1.31	6.73	AMY-3
	8.55	8.90	1.06	1.23	CEL-5
	6.43	6.72	1.09	1.42	CEL-4
Methoxisopropamine	4.93	5.05	1.06	0.59	AMY-1
	6.84	8.16	1.35	7.06	AMY-3
	6.59	7.09	1.15	1.98	CEL-5
	5.33	5.88	1.25	3.10	CEL-4
Deoxymethoxetamine	4.56	4.81	1.16	1.36	AMY-1
	6.71	7.71	1.28	6.19	AMY-3
	8.03	8.10	1.02	0.39	CEL-5
	5.85	6.11	1.09	1.29	CEL-4
2F-2-Oxo-PCE	4.91	5.08	1.09	0.75	AMY-1
	9.83	10.29	1.07	1.76	AMY-3
	10.90	11.04	1.02	0.49	CEL-5
	7.61	8.02	1.09	1.66	CEL-4
(b)
Compound	t1 (min)	t2 (min)	α	Rs	CSP
Ketamine	8.35	9.14	1.15	2.72	AMY-1
	13.88	14.32	1.04	1.16	AMY-3
	13.95	14.36	1.04	0.95	CEL-5
	9.76	10.16	1.06	1.30	CEL-4
Deschloroketamine	8.79	16.51	2.37	17.38	AMY-1
	14.88	32.36	2.49	27.65	AMY-3
	11.68	12.76	1.13	3.13	CEL-5
	8.53	9.15	1.12	2.45	CEL-4
2F-Ketamine	8.81	10.81	1.36	6.15	AMY-1
	16.09	18.52	1.19	5.37	AMY-3
	14.69	15.84	1.10	2.62	CEL-5
	10.13	11.06	1.14	2.92	CEL-4
N-Ethylketamine	5.72	6.23	1.20	2.67	AMY-1
	7.54	8.18	1.15	3.14	AMY-3
	8.57	n.d.	-	-	CEL-5
	6.50	6.78	1.09	1.31	CEL-4
2-Oxo-PCE	5.72	n.d.	-	-	AMY-1
	8.05	8.78	1.15	3.28	AMY-3
	7.17	n.d.	-	-	CEL-5
	5.60	5.79	1.08	0.92	CEL-4
Methoxetamine	6.69	7.16	1.14	1.99	AMY-1
	12.63	13.28	1.07	1.76	AMY-3
	8.71	n.d.	-	-	CEL-5
	6.51	6.69	1.05	0.97	CEL-4
Hydroxetamine	9.56	9.94	1.06	1.07	AMY-1
	15.75	16.85	1.09	2.39	AMY-3
	9.43	9.62	1.03	0.73	CEL-5
	9.13	9.40	1.05	0.86	CEL-4
Methoxpropamine	5.80	5.99	1.07	0.95	AMY-1
	9.19	10.12	1.16	3.59	AMY-3
	7.22	n.d.	-	-	CEL-5
	5.61	5.65	1.01	0.23	CEL-4
Methoxisopropamine	5.10	5.27	1.09	0.87	AMY-1
	7.10	8.47	1.35	6.34	AMY-3
	5.92	6.08	1.06	0.90	CEL-5
	5.02	5.23	1.12	1.29	CEL-4
Deoxymethoxetamine	4.60	4.76	1.11	0.94	AMY-1
	5.65	6.93	1.52	7.30	AMY-3
	6.91	n.d.	-	-	CEL-5
	5.30	5.44	1.07	0.79	CEL-4
2F-2-Oxo-PCE	5.78	6.33	1.21	3.06	AMY-1
	8.21	9.01	1.16	3.55	AMY-3
	8.43	n.d.	-		CEL-5
	6.48	6.68	1.06	1.00	CEL-4

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