Accepted Manuscript Title: Determination of ketamine, norketamine and dehydronorketamine in urine by hollow-fiber liquid-phase microextraction using an essential oil as supported liquid membrane Author: Andr´e Valle de Bairros Rafael Lanaro Rafael Menck de Almeida Mauricio Yonamine PII: DOI: Reference:
S0379-0738(14)00156-X http://dx.doi.org/doi:10.1016/j.forsciint.2014.04.016 FSI 7575
To appear in:
FSI
Received date: Revised date: Accepted date:
21-10-2013 18-3-2014 9-4-2014
Please cite this article as: A.V. Bairros, R. Lanaro, R.M. Almeida, M. Yonamine, Determination of ketamine, norketamine and dehydronorketamine in urine by hollow-fiber liquid-phase microextraction using an essential oil as supported liquid membrane, Forensic Science International (2014), http://dx.doi.org/10.1016/j.forsciint.2014.04.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination
of
ketamine,
norketamine
and
dehydronorketamine in urine by hollow-fiber liquid-phase microextraction using an essential oil as supported liquid
cr
ip t
membrane
André Valle de Bairrosa,*, Rafael Lanarob, Rafael Menck de Almeidaa, Mauricio
us
Yonaminea
Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil
b
Campinas Poison Control Center, Faculty of Medical Sciences, State
M
University of Campinas, Brazil
an
a
* Corresponding author. Tel.: +55 11 30912194; Fax: +55 11 30319055.
te Ac ce p
d
E-mail address: [email protected]; [email protected]
Here, we present a method for the determination of ketamine (KT) and its main metabolites, norketamine (NK) and dehydronorketamine (DHNK) in urine samples by using hollow-fiber liquid-phase microextraction (HF-LPME) in the three-phase mode. The fiber pores were filled with eucalyptus essential oil and a solution of 1.0 mol/L of HCl was introduced into the lumen of the fiber (acceptor phase). The fiber was submersed in the alkalinized urine containing 10% NaCl, and the system was submitted to lateral shaking (2400 rpm) during 30 min. Acceptor phase was withdrawn from the fiber, dried and the residue was then derivatized with trifluoroacetic anhydride (TFAA) for further determination by gas chromatography–mass spectrometry (GC–MS). The 1
Page 1 of 34
calibration curves were linear over the specified range and limits of detection (LoDs) obtained for KT, NK and DHNK were below the cut-off value (1.0 ng/mL) recommended by the United Nations Office on Drugs and Crime (UNODC). A
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totally “green chemistry” approach of the sample extraction was obtained by using essential oil as a supported liquid membrane in HF-LPME. The developed
cr
method was successfully validated and applied to urine samples collected from
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two clinical cases in which KT was suspected to be involved.
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Keywords: Essential oils; Gas chromatography-mass spectrometry; Hollow
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1. Introduction
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d
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fiber-liquid phase microextraction; Ketamine; Urine
Ketamine (KT) is a N-methyl-D-aspartate (NMDA) receptor antagonist
used as anaesthesic in both animals and humans [1]. It was initially abused by medical personnel due to its hallucinogenic properties, and gradually became popular among young user population at dance and rave parties, being one of the recreational drugs known as “club drugs” [2, 3]. KT is an odorless, tasteless and colourless drug and it can be added to beverages, without being perceived by the victim, promoting stupor and sedation together with amnesia. Because of its pharmacological properties, this drug is also misused by offenders in cases of drug-facilitated crimes (DFC) [4-6]. 2
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After administration of KT in humans, this substance is broken down into norketamine (NK), an active metabolite. NK is then dehydrogenated generating dehydronorketamine (DHNK). KT and its main metabolites (NK and DHNK) are
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further transformed by hydroxylation and conjugation prior to elimination (Fig. 1). Approximately 2% is excreted as parent drug, 2% as NK, 16% as DHNK and
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the rest as conjugates of hydroxylated metabolites [4, 7]. According to the
Society of Forensic Toxicologists (SOFT) and United Nations Office on Drugs
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and Crime (UNODC), KT and NK are the target analytes in toxicological
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analysis with suspected involvement of this drug using urine as biological matrix [6, 8]. In spite of the absence of DHNK in the SOFT and UNODC list of targeted
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analytes, this metabolite has also been indicated as a biomarker of administration of KT in the scientific literature [4, 9-11]. In fact, following the
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administration of a single oral dose of KT (50 mg) in six volunteers, KT and NK
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were detected in urine up to 5 and 6 days, respectively, and DHNK could be detected for up to 10 days [12].
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Urine samples continue to be widely used as a biological matrix for the
analysis of psychoactive substances in forensic cases because of the large sample volume that can be collected for analysis, the relative simplicity of the sample preparation and a wider drug detection window compared to blood [6, 13].
Some analytical methods have been described in the scientific literature
for the determination of KT and its main metabolites alone or in combination with other psychoactive substances in urine samples. Gas chromatography– mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) are the main techniques used in the majority of these methods.
3
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Sample preparation techniques used in these chromatographic methods include the conventional liquid-liquid extraction (LLE) [3, 7, 10], solid-phase extraction (SPE) [4, 9, 11-14] and the use of polyvinylidene difluoride (PVDF) filter
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syringes [15]. Miniaturized techniques, such as solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) have been scarcely employed
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for this purpose [2, 16].
Over the last decade, the development of miniaturized extraction
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procedures, especially LPME, has become an encouraging field in analytical
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chemistry. In hollow fiber-liquid phase microextraction (HF-LPME), a water immiscible solvent is immobilized as a thin supported liquid membrane (SLM)
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into the pores of a porous hollow fiber. The lumen of the fiber is filled with acceptor solution and the system is placed in contact with the sample (donor
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phase). The analytes are extracted from the sample (aqueous), through the
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SLM (organic) and finally into the acceptor solution. The acceptor solution can be an organic solvent, providing a two-phase extraction system or it can be an
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aqueous solution, providing a three-phase extraction system. In the three-phase extraction system, analytes must be ionizated, and consequently, trapped in the lumen of the hollow fiber. Due to the high sample-to-acceptor volume ratio, very high analyte enrichments can be obtained by the use of HF-LPME, especially in the three-phase mode. Also, excellent clean-up has been reported from complex biological matrices because the pores size of the fiber provides microfiltration of macromolecules [17, 18]. In the three-phase mode, besides obtaining a better recovery compared to two-phase, this technique allows easy drying and subsequent derivatization of the analytes, a chemical reaction that increases the selectivity and stability of
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the compounds. Therefore, an interesting perspective of three-phase mode is the possibility of performing a totally “green analytical chemistry” by the use of fatty oils or essential oils as SLM instead of using organic solvents. This type of
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extraction can eliminate the use of hazardous organic solvents in sample preparation methods based on HF-LPME [18, 19].
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The aim of the present study was to develop a method for the
determination of KT and its main metabolites (NK and DHNK) in urine samples
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using hollow-fiber liquid phase microextraction (HF-LPME) in three-phase mode
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and gas chromatography-mass spectrometry (GC–MS). The method was fully validated and succesfully applied to urine samples collected from two clinical
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cases, confirming the suspicion of ketamine exposure in these cases.
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2. Experimental
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2.1. Reagents and reference standards
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Ketamine (KT), norketamine (NK) and dehydronorketamine (DHNK)
solutions (1.0 mg/mL) in methanol and the internal standards (IS) ketamine-d4 (KT-d4) and norketamine-d4 (NK-d4) solutions (1.0 mg/mL) also in methanol were purchased from Cerilliant Analytical Reference Standards® (Round Rock, TX, USA). Dihexyl ether, 1-nonanol, undecane, decanol, 1-octanol, xilol, trifluoroacetic anhydride (TFAA) and ethyl acetate were purchased from SigmaAldrich® (MO, USA), while sodium hydroxide and hydrochloric acid were purchased from Merck® (Darmstadt, Germany). Essential oils of canola, clove and peppermint were obtained from Mapric® (São Paulo, SP, Brazil). Essential oil of eucalyptus was purchase from Mapric® (São Paulo, SP, Brazil) and
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Natural Pharma Ltda® (São Paulo, SP, Brazil), while soybean oil and olive oil from Bunge Brazil® (São Paulo, SP, Brazil) were obtained from a local grocery.
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2.2. Preparation of standard solutions Working solutions of KT, NK, DHNK at concentrations of 1.0 µg/mL and
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0.1 µg/mL were prepared with methanol in volumetric glassware. Stock
solutions were stored refrigerated (-20ºC) when not in use. Working solutions of
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the IS (KT-d4 and NK-d4) at a concentration of 1.0 µg/mL were also prepared in
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methanol. 2.3. Instrumentation
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Hollow-fiber Q3/2 Accurel KM polypropylene (600 µm i.d., 200 µm wall thickness and 0.2 µm pore size) was purchased from Membrana (Wuppertal,
d
Germany). Gel-loading pippeter tips Round CC 4853 (0.5 mm; 1-200 µL) were
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purchased from Costar (Corning, NY, USA). Extractions were conducted using
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a multi-tube vortexer model VWR VX-2500 (Thorofare, NJ, USA). The analyses were performed using an Agilent 6850 Network GC System gas chromatograph coupled with an Agilent 5975 Series quadrupole mass seletive detector (MSD) (Wilmington, DE, USA). Samples were injected into the GC-MS by means of an autosampler (Agilent 7693). Injections were made in the splitless mode (2 min and afterwards split vent was turned on in a ratio of 1:50). Chromatographic separation was achieved on a HP-5MS fused-silica capillary column (30 m x 0.25 mm x 0.1 µm film thickness) using helium as the carrier gas at 1.0 mL/min in a constant flow rate mode. The column oven temperature program was as follows: first held at 100°C, then programmed at 10°C/min to 200°C (hold 1 min), then 5°C/min at 210°C (hold 1 min); 40°C/min at 300°C (hold 1 min). 6
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Injection port and transfer line were 260°C and 280°C respectively. The MS was operated by electron ionization (70 eV) in selected ion monitoring (SIM) mode. The following ions were chosen for SIM analyses (quantification ions
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underlined): KT: 270, 262, 305; KT-d4: 274, 266; NK: 284, 256, 214; NK-d4: 288, 260; DHNK: 214, 282, 317. NK-d4 was also used as IS for DHNK.
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2.4. Urine samples
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The drug-free urine samples were obtained from nonuser volunteers (laboratory staff) and were stored at –20°C until analysis. The drug-free
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samples were used for the validation of the analytical method. The urine samples collected from real cases were obtained from the Campinas Poison
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Control Center, Faculty of Medical Sciences, State University of Campinas, Brazil. The protocol of study was previously approved by the Faculty of
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Pharmaceutical Sciences Ethics Committee, University of São Paulo, Brazil
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(Ethics Protocol Approval n° CEP 98156).
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2.5. Sample preparation
An aliquot of 2.0 mL of urine was transferred into a 4 mL glass tube,
followed by the addition of 20 ng of each IS (KT-d4 and NK-d4). The sample was alkalinized with NaOH 2 mol/L solution until pH 10 and added 10% NaCl (m/v). A 7.5-cm hollow fiber was filled-up with eucalyptus oil in its pores. The fiber lumen was filled with 18 µL of HCl 1.0 mol/L using a gel-loading pipetter tip and the ends of the hollow-fiber were sealed by mechanical pressure with pliers. The fiber was then introduced as U-shape into the sample solution. During the extraction, the assembly was submitted for shaking at 2400 rpm for 30 minutes in the multi-tube vortexer. After extraction, the acceptor phase was 7
Page 7 of 34
withdrawn from the fiber and dried under nitrogen stream at 40ºC. The residue was derivatized with 100 µL of TFAA: ethyl acetate (1:1) at 60ºC for 30 min. After cooling, the samples were dried once more (40ºC under nitrogen stream)
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and resuspended with 35 µL of ethyl acetate. An aliquot of 2.0 µL of this solution was injected into the GC–MS system.
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2.6. Optimization of the method
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The study of optimization of the method was performed taking into consideration the choice of SLM, pH of donor phase, influence of acceptor
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phase, time for extraction, intensity of shaking and addition of salt to the extraction yield. Fortified urine samples at a concentration of 15 ng/mL of each
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analyte were submitted to the method previously described. The efficiency of extraction was evaluated by the absolute area produced by each analyte in all
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tested conditions. The following parameters were studied: SLM (dihexyl ether,
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xilol, 1-nonanol, decanol, 1-octanol, undecane, clove oil, eucalyptus oil,
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peppermint oil, canola oil, soybean oil and olive oil); pH of donor phase (pH 9, 10, 11, 12 and 13); acceptor phase (0.010, 0.025, 0.050, 0.075, 0.10 and 1.0 mol/L HCl); time for extraction (5, 10, 15, 20, 30 and 45 min); agitation (800, 1200, 1600, 2000 and 2400 rpm). The salting out effect was also tested by adding 0, 5, 10 and 20 % of NaCl (m/v) to the sample before extraction. All the optimization procedure was performed in three replicates for each parameter evaluated. The other fixed parameters set as constant while changing a given variable were those described in section 2.5 (Sample preparation). 2.7. Validation of the method
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The validation of the method was carried out by establishing both limit of detection (LoD) and quantitation (LoQ), selectivity/specificity, robustness, linearity, intra and inter-assay accuracy, precision, recovery, and dilution
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integrity after optimization of the method. All validation procedures were performed in six replicates for each parameter evaluated. Specific topics about
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validation of the method are described below.
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2.7.1. Limit of detection (LoD) and limit of quantification (LoQ)
The limit of blank (LoB) was established to ensure a reliable LoD. The
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LoB is the highest apparent concentration expected to be found when 60 replicates of 20 different blank samples containing no analytes are tested. The
[20].
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blank)
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data are expressed in the equation LoB = mean blank + 1.645 (standard deviation
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The LoD can be defined as the lowest concentration of analyte in a sample which can be reliably distinguished from the LoB and at which detection
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is feasible. The procedure to determinate the LoD was similar to how the LoB was determined, by using low concentrations of the analyte spiked into negative urine samples and using the equation LoD = LoB + 1.645 (standard deviation low concentration sample).
When it is used 1.645 standard deviation, no more than 5% of
the values should be less than the LoB. If the observed LoD sample values meet this criterion, the LoD is considered established or verified. The LoQ can be defined as the lowest concentration of a sample that can still be quantified with acceptable precision and accuracy. The acceptance criteria were values < 20% (RSD) for precision and accuracy between 80-120% [20, 21]. 2.7.2. Interference studies 9
Page 9 of 34
Interference studies were evaluated by assaying ten different drug-free urine samples and 10 potential interfering drug compounds. Urine samples were extracted and analyzed according to the previously described procedure
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for assessment of endogenous substances. Peaks at the retention time of interest were compared to those from urine samples spiked with the analytes at
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the LOQ. The method was also evaluated for potential interfering substances through the analysis of blank samples fortified with 1000 ng/mL of methamphetamine,
benzoylecgonine,
11-nor-9-carboxy-
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amphetamine,
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tetrahydrocannabinol, diazepam, morphine, codeine, acetylsalicylic acid, naphazoline and caffeine. Acceptance criteria for this assay was the absence of
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interfering substances at the retention times of the analyte(s) of interest and of their respective IS [6, 21].
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2.7.3. Robustness
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Robustness is a measure of the ability of a method to remain unaffected
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by small changes that might occur during analysis. In this case, possible differences in the analysis by using essential oils of eucalyptus deriving from different manufacturers were evaluated. Two sets of urine samples containing known concentrations of 1.5, 12.5 and 40.0 ng/mL of all analytes (six replicates each) were analysed according to the method described in the section 2.5 (Sample preparation). In one set, eucalyptus oil was deriving from Mapric® (São Paulo, SP, Brazil). In another set, eucalyptus oil was deriving from Natural Pharma Ltda® (São Paulo, SP, Brazil). 2.7.4. Linearity
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Linearity was established by analyzing urine samples containing all the analytes of interest at the following concentrations: 0.5, 1, 2.5, 5, 10, 25 and 50 ng/ml of KT, NK and DHNK. Six replicate samples were analyzed at each
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concentration level.
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2.7.5. Precision and accuracy
The precision and accuracy study was performed by analyzing urine
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samples containing known concentrations of 1.5, 12.5 and 40 ng/mL of KT, NK and DHNK during three consecutive days. The analyses were performed in six
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replicates for each day. Precision, defined as the relative standard deviation (RSD), was determined by intra and inter-day repetitions. Experimental
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concentrations were obtained using the standard calibration curves. Accuracy was expressed as a percentage of the known concentration, i.e., mean
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2.7.6. Recovery
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measured concentration/nominal concentration × 100.
The recovery studies were performed by preparing two sets of urine
samples. One set of samples (set A), consisting of three concentrations (1.5, 12.5 and 40 ng/mL) for each analyte, were analyzed in six replicates for each concentration according to the method described in Section 2.5 (Sample preparation). The second set (set B), also comprised samples in six replicates for each concentration (1.5, 12.5 and 40 ng/mL). However, for set B, the analytes were spiked into the samples immediately after LPME procedure. Absolute recovery was evaluated by comparison of the mean response obtained for the set A (processed) and the response of set B (unprocessed). The unprocessed response represented 100% recovery. 11
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2.7.7. Dilution integrity Dilution integrity is a parameter that allows evaluating samples with levels of analytes above the calibration curve. When dilution integrity is
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necessary, it should be demonstrated by spiking the matrix with analytes above the highest concentration of the curve and then diluting this sample with blank
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should be within the set criteria, i.e. within ±15% [22].
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matrix (at least five determinations per dilution factor). Accuracy and precision
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2.8. Aplication to real cases
To prove the applicability of this analytical method, real cases that
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involved ketamine exposure were analyzed. The two cases corresponded to two women (35 and 27 years old) who misused ketamine as a suicide attempt
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or for supposed criminal purposes. Urine samples were collected at the hospital
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in all the cases. The quantification was based on the ratios of the ion peak areas of the compounds to the IS ion peak areas. The calibration cures were
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used to determine both ketamine and its metabolite concentrations.
3. Results and discussion 3.1. Sample preparation
The majority of methods developed to detect ketamine and metabolites in urine samples comprise of conventional liquid-liquid extraction (LLE) or solid phase extraction (SPE) as sample preparation techniques, before chromatographic analysis. However, these techniques require relatively large volumes of organic solvents that are toxic to the analyst and hazardous to the environment. In fact, 12
Page 12 of 34
the so-called “green chemistry” has gained considerable interest in a broad field of the analytical area, because it reduces exposure and/or waste of toxic agents from sample preparation. For this reason, miniaturized techniques, such as
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SPME and LPME, have adopted this philosophy and use little or no organic solvent in their procedures [19].
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In the scientific literature, only two recent publications considered the
utilization of SPME or LPME for the analysis of ketamine and its metabolites in
chromatographic–mass
spectrometric
(SPME-GC-MS)
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microextraction-gas
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urine [2, 16]. Brown et al [2] developed and validated a solid-phase
method for the determination of four “club drugs” (gamma-hydroxybutyrate,
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ketamine, methamphetamine and methylenedioxymethamphetamine) in human urine. However, SPME presents some disadvantages: it is expensive, the
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fragile fiber has a limited lifetime and due to the high cost, there is the possibility
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of sample carry-over effect because the same fiber may be used for multiple extractions. LPME, on the other hand, has eliminated the carry-over effect since
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the hollow fibers can be discarded after each extraction due to their low cost [18].
Xiong
et
al
[16]
developed
a
HF-LPME
combined
with
gas
chromatography-flame ionization detection (GC-FID) for the simultaneous quantification
of
amphetamine,
methamphetamine,
methylenedioxyamphetamine, methylenedioxymethamphetamine, caffeine and ketamine in drug abuser urine samples. The LoD obtained in their study for ketamine was 8.0 ng/mL. In the present work, the LoDs obtained for KT, NK and DHNK were 0.25, 0.1 and 0.1 ng/mL, respectively. Depending on the configuration used, there is a limitation related to the number of samples that can be processed in the same batch. Here we used the
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Page 13 of 34
U-shape configuration without any supporters connecting the hollow-fiber ends. Due to the simplicity of these LPME units, many samples can be processed at the same time, providing a high sample throughput, similar to others works [23,
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24]. The major disadvantage is the lack of automation of the process since it is a relatively new technique.
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Despite the additional step in the sample preparation and the toxicity of the TFAA, derivatization was required for the GC-MS analysis. Without the use
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of derivatizing reagents, overlapping of the chromatographic peaks between
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underivatized KT and DHNK was observed. Trifluoroacetic anhydride (TFAA), a low cost and commonly used acylating reagent, showed to be the best choice
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as a derivatization agent for KT and its metabolites [14]. It allowed the detection of low levels of KT and its metabolites with an excellent chromatographic
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separation among the analytes. Mass spectra of the derivatizated KT, NK and
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DHNK obtained in full scan mode are shown in Fig. 2. In summary, at least 40 urine samples could be processed in less than 3
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hrs by one analyst alone, considering the entire procedure from sample preparation until injection into the chromatographic system. 3.2. Optimization
For the optimization of the method, different pHs of the donor phase
(sample solution) and the acceptor phase were tested. KT, NK and DHNK are basic drugs and urine samples were adjusted from pH 9 to13 using a solution of 2 mol/L NaOH. The best pH of urine sample for the extraction of these analytes was 10, but a decrease in the signal for DHNK was observed at pH values > 11, similar to the findings of Sams and Pizzo [25]. Probably this behavior occurs due to the alpha-beta unsaturated ketone present in the cyclohexenone ring of 14
Page 14 of 34
the DHNK. In this condition, NaOH acts as nucleophile which reacts in the betacarbon and generates enolate, an intermediate ion stabilizated by resonance between negative charge of the alpha-carbon and carbonyl group [26]. This
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particular aspect of DHNK could have an influence on the low capacity of extraction of this compound at pH > 11. Taking into consideration the acceptor
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phase, a concentration of HCl greater than 0.025 mol/L slightly increased the
response. However, concentrations greater than 1.0 mol/L produced a
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dramatically reduction of the peak area of KT and irregular responses for NK
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and DHNK. Probably the high concentration of acid solution caused degradation of these molecules. Therefore, HCl 1.0 mol/L was chosen as
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acceptor phase.
Different organic solvents were also tested as SLM for the LPME system.
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The best organic solvent for the analytes was dihexyl ether. Essential and fatty
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oils were evaluated in comparison to dihexyl ether. Dihexyl ether was the best organic solvent for extraction of KT, while NK and DHNK were better extracted
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using essential oil of eucalyptus (Fig. 3). Therefore, essential oil of eucalyptus was chosen to be used as SLM in the present method. By replacing the organic solvent for an essential oil, it was possible to obtain a totally “green chemistry” approach for the extration procedure in the present method. The equilibrium of analytes between donor phase and acceptor phase
can be established more rapidly with the agitation of LPME system. In this work, the effect of agitation velocity on the extraction yield of the analytes was investigated by shaking the LPME system at different agitation rates (800, 1200, 1600, 2000 and 2400 rpm) by using a multi-tube vortexer. The experimental results showed that the peak areas of KT, NK and DHNK were increased at
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2400 rpm. Subsequently, an agitation rate of 2400 rpm was chosen for further studies. Extraction time using HF-LPME for KT, NK and DHNK started from 5 to
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45 min. Maximum peak area for the analytes were obtained with 30 min of agitation. Surprisingly, at 45 min, all analytes presented a decrease in the
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response. Therefore, 30 min was selected as ideal time for extraction. Moreover, salting-out effect was evaluated by increasing NaCl concentration
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from 0 to 20% (m/v) in sample solution. The concentration of 10% of salt
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provided higher peak area and better chromatograms. 3.3. Validation of the method
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The validation of the method was carried out by establishing selectivity/specificity, robustness, linearity, recovery, intra and inter-day
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precision, accuracy, limit of detection (LoD), limit of quantification (LoQ) and
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dilution integrity. No interfering peaks due to endogenous or exogenous
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substances were observed at the retention time of the compounds of interest. The robustness tests results showed differences less than 20% (83.4–112.1%) were observed between samples analyzed by using eucalyptus oils from two different suppliers in three levels of concentration. Other confidence parameters of the validated method are summarized in the Table 1. After optimization of the extraction, the recovery values for the study of
KT, NK and DHNK ranged from 64.6 to 101%. These results can be considered excellent since recoveries exceeding 80–90% are rare due to analyte trapping within the SLM in the three-phase mode of LPME [18]. For quantification by HFLPME, the use of deuterated labelled IS for each analyte is recommended to improve the precision of the method. In this work, KT-d4 and NK-d4 were used 16
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as IS for their respective analogues and NK-d4 was also used as IS for DHNK, since the deuterated analogue for this substance is not commercially available. Nevertheless, precision was considered acceptable for all tested substances
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(RSD < 13.5% for intra-day precision). The calibration curves were linear over the specified range (0.5–50 ng/mL). The linear regression equations and the
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respective relative standard deviation (RSD) were: Y= 0.1103X+0.0032, r2= 0.9996 (KT); Y= 0.1022X+0.0513, r2 = 0.9985 (NK), and Y= 0.084X+0.1149, r2=
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0.9936 (DHNK), where Y and X represent the relationship between the peak
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area ratio (compound/IS) and the corresponding calibration concentrations. The values of accuracy for these analytes at three different concentration levels
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ranged from 86.2 to 95.9%.
The LoD and LoQ obtained for the analytes were below the minimum
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required performance limit - MRPL (1.0 ng/mL for ketamine and norketamine)
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established by UNODC [6]. This limit was set taking into consideration cases of drug-facilitated sexual assault (DFSA). DFSA incidents are generally reported
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later than 24h after the alleged assault, when very little drug still remains in the victim’s body. A summary of developed methods to detect ketamine and its metabolites in urine samples is shown in Table 2. As it can be observed, the LoQs obtained in the present method are lower than those reported by the references [2-4, 7, 10-16, 27-30] and higher than the ones reported in the reference [9], who worked with UPLC-MS/MS. Only a few studies consider the use of hydrolysis procedures or the detection of hydroxylated compounds for the analysis of KT and metabolites in urine samples [7, 31] (Table 2). Lin and Lua [7] analyzed 24 paired urine samples (with and without hydrolysis procedure) and they obtained the
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Page 17 of 34
hydrolyzed and unhydrolyzed ratio for each analyte. The obtained H/U ratios were in the range of 0.85-1.85, 1.05-2.22 and 0.80-1.87 for KT, NK and DHNK, respectively. However, hydrolysis step is an additional and lengthy process, and
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our method showed that hydrolytic cleavage was not necessary to achieve the recommended maximum detection limit (1.0 ng/mL) established by UNODC for
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the targeted analytes (KT and NK) [6, 8] and also for DHNK.
Due to the possibility of urine samples with concentrations of KT, NK and
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DHNK above the calibration range, the study of the dilution integrity was done
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to ensure that the method is precise and accurate after this procedure. Dilution integrity was performed by spiking the blank urine with 350 and 3500 ng/mL and
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diluting this sample with the same matrix in 10 and 100 times (six replicates per dilution factor). Accuracy of these dilutions for KT, NK and DHNK ranged from
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86.20 to 95.90%, while the precision (RSD) was below 10.30%. These results
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are in accordance with criteria established by EMA (RSD + 15%) [22].
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3.4. Application of the method
The developed HF-LPME and GC-MS method was applied for the
quantification of KT and its metabolites in two real cases handled by the Campinas Poison Control Center (SP-Brazil). These cases are described below:
Case 1: a 35 year-old woman disappeared from her workplace for 10
days and she was found unconscious in a car. When she woke up, the woman said she had been kidnapped, but she disappeared again and she was later found unconscious in a bathroom of shopping mall. The patient was admitted to the hospital with Glasgow coma scale 10, 120/70 mmHg of blood pressure and
18
Page 18 of 34
normal respiratory rate. She remained in the intensive care unit during 24 hours and reported her suicide attempt. Case 2: a 27 year-old woman was found in a sugar-cane plantation with
ip t
burns in her body and unstable neurological status, alternating between periods of confusion and lucidity. When the patient arrived at the hospital, her blood test
cr
showed leukocytosis and hypernatremia, while other blood exams showed normal values. A urine sample was collected 2 hours after admittance in the
us
hospital. First, she was suspected of attempted rape, but the gynecologist
an
dismissed this hypothesis.The patient was then discharged from the hospital after an 8-days stay.
M
KT, NK and DHNK were detected in both urine samples from the two cases and the results are presented in the Table 3. In the case 1, where
d
concentrations were above the highest level of the calibration curve, samples
te
were diluted appropriately with blank urine to fit into the calibration curve. Relatively high levels of ketamine and its metabolites were found in the urine
Ac ce p
sample and they are in accordance with the case history (suicide attempt). In the case 2, in spite of the gynecologist dismissed the hypothesis of attempted rape, the possibility of drug-facilitated crime should not be ignored. Concentrations of ketamine and norketamine were a litlle higher than the minimum required performance limit - MRPL (1.0 ng/mL, according to the UNODC guidelines) [6]. Fig. 4 shows chromatograms obtained with the practical use of this method for the analyses of urine samples.
4. Conclusion
19
Page 19 of 34
The results showed that the HF–LPME/GC-MS method is well suited for the determination of ketamine and its main metabolites in human urine samples. Compared to other methods, it revealed some practical advantages such as
ip t
simple device, relatively low cost and sensitivity. In addition, no organic solvent was required for the extraction as an essential oil (eucalyptus oil) was used as a
cr
SLM, providing a green bioanalytical method. The method developed in this
us
study can be useful in the fields of both clinical and forensic toxicology.
an
5. References
[1] E. Visser, S.A. Schug, The role of ketamine in pain management, Biomed.
M
Pharmacother. 60 (2006) 341-348.
[2] S.D. Brown, D.J. Rhodes, B.J. Pritchard, A validated SPME-GC-MS method
te
171 (2007) 142-150.
d
for simultaneous quantification of club drugs in human urine, Forensic Sci.Int.
Ac ce p
[3] K. Lian, P. Zhang, L. Niu, D. Bi, S. Liu, L. Jiang, W. Kang, A novel derivatization approach for determination of ketamine in urine and plasma by gas chromatography-mass spectrometry, J.Chromatogr. A 16 (2012) 104109.
[4] E.M. Kim, J.S. Lee,S.K. Choi, M.A. Lim, H.S. Chung, 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), Forensic Sci. Int. 174 (2008) 197-202. [5] M.K. Shbair, S. Eljabour, M. Lhermitte, Drugs involved in drug-facilitated crimes: part I: alcohol, sedative-hypnotic drugs, gamma-hydroxybutyrate and ketamine. A review, Ann. Pharm. Fr. 68 (2010) 275-285. 20
Page 20 of 34
[6] United Nations Office on Drugs and Crime (UNODC). Guidelines for the forensic analysis of drug facilitating sexual assault and other criminal acts 2011.
United
Nations,
New
York,
2011.
Available
from:
ip t
http://www.unodc.org/documents/scientific/forensic_analys_of_drugs_facilitati ng_sexual_assault_and_other_criminal_acts.pdf. (accessed 15/05/ 2013).
cr
[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.
us
28 (2004) 181-186.
an
[8] Society of Forensic Toxicologists (SOFT). Recommended minimum performance limits for common DFSA drugs and metabolites in urine
M
samples. United States of America, Mesa, 2012. Available from: http://softtox.org/sites/default/files/SOFT_DFSA_Rec_Det_Limits_3-2012.pdf.
d
(accessed 15/05/ 2013).
te
[9] M.K. Huang, C. Liu, J.H. Li, S.D. Huang, Quantitative detection of ketamine, norketamine, and dehydronorketamine in urine using chemical derivatization
Ac ce p
followed by gas chromatography-mass spectrometry, J. Chromatogr. B. 820 (2005) 165-173.
[10] P.S. Cheng, C.Y. Fu, C.H. Lee, C. Liu, C.S. Chien, GC-MS quantification of ketamine, norketamine, and dehydronorketamine in urine specimens and comparative study using ELISA as the preliminary test methodology, J. Chromatogr. B 852 (2007) 443-449.
[11] C.Y. Chen, M.R. Lee, F.C. Cheng, G.J. Wu,Determination of ketamine and metabolites in urine by liquid chromatography-mass spectrometry, Talanta 72 (2007) 1217-1222.
21
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[12] M.C. Parkin, S.C. Turfus, N.W. Smith, J.M. Halket, R.A. Braithwaite, S.P. Elliott, M.D. Osselton, D.A. Cowan, A.T. Kicman ,Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem
ip t
mass spectrometry, J. Chromatogr. B. 876 (2008) 137-142. [13] N. Harun, R.A Anderson, E.I. Miller, Validation of an enzyme-linked
cr
immunosorbent assay screening method and a liquid chromatographytandem mass spectrometry confirmation method for the identification and
us
quantification of ketamine and norketamine in urine samples from Malaysia,
an
J. Anal. Toxicol. 33 (2009) 310-321.
[14] H.R. Lin, A.C. Lua, Simultaneous determination of amphetamines and
M
ketamines in urine by gas chromatography/mass spectrometry, Rapid Commun. Mass Spectrom. 20 (2006) 1724-1730.
d
[15] T. Nema, E.C. Chan, P.C. Ho, Extraction of ketamine from urine using a
te
miniature silica monolithic cartridge followed by quantification with liquid chromatography tandem mass spectrometry (LC-MS/MS), J. Sep. Sci. 34
Ac ce p
(2011) 1041-1046.
[16] J. Xiong, J.Chen, M.He, B. Hu, Simultaneous quantification of amphetamines, caffeine and ketamine in urine by hollow fiber liquid phase microextraction
combined
with
gas
chromatography-flame
ionization
detector, Talanta 82 (2010) 969-975.
[17] J. Lee, H.K. Lee, K.E. Rasmussen, S. Pedersen-Bjergaard, Environmental and bioanalytical applications of hollow fiber membrane liquid-phase microextraction: a review, Anal. Chim. Acta. 624 (2008) 253-268.
22
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[18] S. Pedersen-Bjergaard, K.E. Rasmussen, Liquid-phase microextraction with porous hollow fibers, a miniaturized and highly flexible format for liquidliquid extraction,J. Chromatogr. A. 1184 (2008) 132-142.
ip t
[19] S. Pedersen-Bjergaard, K.E. Rasmussen, Liquid-phase microextraction utilising plant oils as intermediate extraction medium--towards elimination of
cr
synthetic organic solvents in sample preparation, J. Sep. Sci. 27 (2004) 1511-1516.
us
[20] D.A. Armbruster, T. Pry, Limit of blank, limit of detection and limit of
an
quantitation, Clin. Biochem. Rev.29 (2008) S49-52.
[21] F.T. Peters, O.H. Drummer, F. Musshoff, Validation of new methods,
M
Forensic Sci. Int. 165 (2007) 216-224.
[22] European Medicine Agency (EMEA). Guideline on bioanalytical method
d
validation. United Kingdom, London, 2011.
Muñoz,
M.D.
te
[23] R.A. Menck, D.S.D. Lima, S.C. Seulin, V. Leyton, C.A. Pasqualucci, D.R. Osselton,
M.
Yonamine,
Hollow-fiber
liquid-phase
Ac ce p
microextraction and gas chromatography-mass spectrometry of barbiturates in whole blood samples, J. Sep. Sci. 35 (2012) 3361-3368.
[24] L. N. Pantaleao, B.A.B. Paranhos, M. Yonamine, Hollow-fiber liquid-phase microextraction of amphetamine-type stimulants in human hair samples, J. Chromatogr. A. 7 (2012) 1-7.
[25] R. Sams, P. Pizzo, Detection and identification of ketamine and its metabolites in horse urine, J. Anal. Toxicol. 11 (1987) 58-62. [26] Aldehydes and ketones: nucleophilic addition reactions, in: J. Mc Murry, Organic Chemistry – 6th edition, Cengage Learning, Stamford, 2005, pp. 704705.
23
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[27] H.H. Lee, J.F. Lee, S.Y. Lin, P.H. Chen, B.H. Chen, Simultaneous determination of HFBA-derivatized amphetamines and ketamines in urine by gas chromatography-mass spectrometry, J. Anal. Toxicol.35 (2011) 162-169.
ip t
[28] M.M.R. Fernandez, M. Laloup, M. Wood, G. De Boeck, M. Lopez-Rivadulla, P. Wallemacq, N. Samyn, Liquid chromatography-tandem mass spectrometry for
the
simultaneous
analysis
of
multiple
hallucinogens,
cr
method
chlorpheniramine, ketamine, ritalinic acid, and metabolites, in urine, J. Anal.
us
Toxicol. 31 (2007) 497-504.
an
[29] Y. Fan, Y.Q. Feng, S.L. Da, X.P. Gao, In-tube solid-phase microextraction with poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary
M
for direct high-performance liquid chromatographic determination of ketamine in urine samples, Analyst 129 (2004) 1065-1069.
d
[30] S.L. Chou, M.H. Yang, Y.C. Ling, Y.S. Giang, Gas chromatography-isotope
te
dilution mass spectrometry preceded by liquid-liquid extraction and chemical derivatization for the determination of ketamine and norketamine in urine, J.
Ac ce p
Chromatogr. B799 (2004) 37-50.
[31] R. Moaddel, S.L. Venkata, M.J. Tanga, J.E. Bupp, C.E. Green, L. Iyer, A. Furimsky, M.E. Goldberg, M.C. Torjman, I.W. Wainer, A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome, Talanta 82 (2010) 1892-1904.
Legends Figure 1. Chemical structure of target drug analytes.
24
Page 24 of 34
Figure 2. Mass spectra in full scan mode and chemical structure of TFAA derivatives of ketamine, norketamine and dehydronorketamine; A) Ketamine; B) Norketamine; C) Dehydronorketamine. Figure 3. Bar graphs showing efficiencies of ketamine, norketamine and
ip t
dehydronorketamine with the use of six different supported liquid membranes for the hollow fiber–liquid phase microextraction procedure. SLM, supported liquid membrane.
cr
Figure 4. Chromatogram obtained with the analysis of ketamine in urine using
HF-LPME. (A) blank urine sample; (B) urine sample spiked at 10.0 ng/mL of KT,
us
KT-D4, NK, NK-D4 and DHNK; (C) real urine sample containing 7.3, 5.3 and 6.8 ng/mL of KT, NK and DHNK respectivaly. KT, ketamine; KT-D4, ketamineNK,
norketamine;
NK-D4,
norketamine-deuterated;
DHNK,
an
deuterated;
Ac ce p
te
d
M
dehydronorketamine.
25
Page 25 of 34
Table 1. Confidence parameters of the validated method for the determination of ketamine, norketamine and dehydronorketamine in urine samples (six replicates for each point). Confidence parameters
KT
NK
C1
101.0
94.3
C2
91.1
88.5
C3
85.2
86.9
LoD (ng/mL)
0.25
0.10
LoQ (ng/mL)
0.50
DHNK
C2
10.1
C3
cr
68.1
us an
7.5
0.50
64.6
0.10 0.50
9.2
9.3
6.7
13.5
2.9
4.7
9.9
9.1
6.4
16.9
7.3
5.9
9.8
3.0
3.6
14.1
108.0
105.4
118.9
88.3
92.0
106.3
99.0
94.9
111.7
10 times
0.9
1.7
7.0
100 times
2.0
8.0
10.3
10 times
95.9
95.4
86.2
100 times
92.6
93.3
88.7
M
C1
69.7
d
Intra-day precision (%RSD)
ip t
Recovery (%)
Inter-day precision (%RSD)
te
C1 C2
Ac ce p
C3 Accuracy (%) C1 C2 C3
Dilution integrity
Precision (%RSD)
Accuracy (%)
26
Page 26 of 34
C1, 1.5 ng/mL; C2, 12.5 ng/mL; C3, 40 ng/mL; 10 times, 350 ng/mL; 100 times, 3500 ng/mL; LoD, limit of detection; LoQ, limit of quantification; Precision and accuracy was performed during three consecutive days; %RSD, relative standard deviation; KT,
Ac ce p
te
d
M
an
us
cr
ip t
ketamine; NK, norketamine; DHNK, dehydronorketamine.
27
Page 27 of 34
Table 2. Summary of analytical methods for the determination of KT and its
Extraction
Detection
LoQ (ng/mL)
Reference
KT
SPME
GC-MS
500
2
KT
LLE
GC-MS
10
3
KT and NK
SPE
GC-PCI/MS
50
KT and NK
LLE
GC-MS
5 and 10
7
KT and NK
SPE
UPLC-MS/MS
0.10
9
KT, NK and DHNK
LLE
GC-MS
20, 20 and 30
10
KT
SPE
LC-MS/MS
1.60
11
KT, NK and DHNK
SPE
GC-MS
25, 30 and 50
12
KT, NK and DHNK
SPE
GC-MS
1.5
13
KT, NK and DHNK
SPE
GC-MS
15, 10 and 20
14
KT, NK and DHNK
PVDF filter
LC-APCI/MS
3.17, 1.60 and 1.10
15
KT
HF-LPME
GC-FID
30
16
KT and NK
SPE
LC-MS/MS
0.50 and 20
27
KT
SPME
LC-UV
21.30
28
KT and NK
LLE
GC-MS
13 and 9
29
KT and NK
SPE
MECK
5
30
cr
us
an
d
te
ip t
Analytes
M
major metabolites in urine samples.
4
SPME, solid phase microextration; LLE, liquid-liquid extraction; SPE, solid
Ac ce p
phase extraction; PVDF, polyvinylidene difluoride; HF-LPME, hollow fiber-liquid phase microextraction; GC-MS, gas chromatography-mass spectrometry; PCI, positive chemical ionization; LC-UV, liquid chromatography-ultraviolet; FID, flame ionization detector; UPLC, ultra performance liquid chromatography; MECK, micellar electrokinetic chromatography
28
Page 28 of 34
Table 3. Results of the analysis of urine samples from real cases. Urine samples
KT (ng/mL)
NK (ng/mL)
DHNK (ng/mL)
Case 1
87.3
5805
8760
Case 2 7.3 5.3 KT, ketamine; NK, norketamine; DHNK, dehydronorketamine.
Ac ce p
te
d
M
an
us
cr
ip t
6.8
29
Page 29 of 34
Acknowledgments
Acknowledgments Financial supports from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e
Ac
ce pt
ed
M
an
us
cr
ip t
Tecnológico (CNPq) are gratefully acknowledged.
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Ac
ce
pt
ed
M
an
us
cr
i
Figure 1
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te
d
M
an
us
cr
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Figure 2
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Ac
ce
pt
ed
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an
us
cr
i
Figure 3
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Ac
ce
pt
ed
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an
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i
Figure 4
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S0379-0738(14)00156-X http://dx.doi.org/doi:10.1016/j.forsciint.2014.04.016 FSI 7575
To appear in:
FSI
Received date: Revised date: Accepted date:
21-10-2013 18-3-2014 9-4-2014
Please cite this article as: A.V. Bairros, R. Lanaro, R.M. Almeida, M. Yonamine, Determination of ketamine, norketamine and dehydronorketamine in urine by hollow-fiber liquid-phase microextraction using an essential oil as supported liquid membrane, Forensic Science International (2014), http://dx.doi.org/10.1016/j.forsciint.2014.04.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination
of
ketamine,
norketamine
and
dehydronorketamine in urine by hollow-fiber liquid-phase microextraction using an essential oil as supported liquid
cr
ip t
membrane
André Valle de Bairrosa,*, Rafael Lanarob, Rafael Menck de Almeidaa, Mauricio
us
Yonaminea
Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil
b
Campinas Poison Control Center, Faculty of Medical Sciences, State
M
University of Campinas, Brazil
an
a
* Corresponding author. Tel.: +55 11 30912194; Fax: +55 11 30319055.
te Ac ce p
d
E-mail address: [email protected]; [email protected]
Here, we present a method for the determination of ketamine (KT) and its main metabolites, norketamine (NK) and dehydronorketamine (DHNK) in urine samples by using hollow-fiber liquid-phase microextraction (HF-LPME) in the three-phase mode. The fiber pores were filled with eucalyptus essential oil and a solution of 1.0 mol/L of HCl was introduced into the lumen of the fiber (acceptor phase). The fiber was submersed in the alkalinized urine containing 10% NaCl, and the system was submitted to lateral shaking (2400 rpm) during 30 min. Acceptor phase was withdrawn from the fiber, dried and the residue was then derivatized with trifluoroacetic anhydride (TFAA) for further determination by gas chromatography–mass spectrometry (GC–MS). The 1
Page 1 of 34
calibration curves were linear over the specified range and limits of detection (LoDs) obtained for KT, NK and DHNK were below the cut-off value (1.0 ng/mL) recommended by the United Nations Office on Drugs and Crime (UNODC). A
ip t
totally “green chemistry” approach of the sample extraction was obtained by using essential oil as a supported liquid membrane in HF-LPME. The developed
cr
method was successfully validated and applied to urine samples collected from
us
two clinical cases in which KT was suspected to be involved.
an
Keywords: Essential oils; Gas chromatography-mass spectrometry; Hollow
Ac ce p
1. Introduction
te
d
M
fiber-liquid phase microextraction; Ketamine; Urine
Ketamine (KT) is a N-methyl-D-aspartate (NMDA) receptor antagonist
used as anaesthesic in both animals and humans [1]. It was initially abused by medical personnel due to its hallucinogenic properties, and gradually became popular among young user population at dance and rave parties, being one of the recreational drugs known as “club drugs” [2, 3]. KT is an odorless, tasteless and colourless drug and it can be added to beverages, without being perceived by the victim, promoting stupor and sedation together with amnesia. Because of its pharmacological properties, this drug is also misused by offenders in cases of drug-facilitated crimes (DFC) [4-6]. 2
Page 2 of 34
After administration of KT in humans, this substance is broken down into norketamine (NK), an active metabolite. NK is then dehydrogenated generating dehydronorketamine (DHNK). KT and its main metabolites (NK and DHNK) are
ip t
further transformed by hydroxylation and conjugation prior to elimination (Fig. 1). Approximately 2% is excreted as parent drug, 2% as NK, 16% as DHNK and
cr
the rest as conjugates of hydroxylated metabolites [4, 7]. According to the
Society of Forensic Toxicologists (SOFT) and United Nations Office on Drugs
us
and Crime (UNODC), KT and NK are the target analytes in toxicological
an
analysis with suspected involvement of this drug using urine as biological matrix [6, 8]. In spite of the absence of DHNK in the SOFT and UNODC list of targeted
M
analytes, this metabolite has also been indicated as a biomarker of administration of KT in the scientific literature [4, 9-11]. In fact, following the
d
administration of a single oral dose of KT (50 mg) in six volunteers, KT and NK
te
were detected in urine up to 5 and 6 days, respectively, and DHNK could be detected for up to 10 days [12].
Ac ce p
Urine samples continue to be widely used as a biological matrix for the
analysis of psychoactive substances in forensic cases because of the large sample volume that can be collected for analysis, the relative simplicity of the sample preparation and a wider drug detection window compared to blood [6, 13].
Some analytical methods have been described in the scientific literature
for the determination of KT and its main metabolites alone or in combination with other psychoactive substances in urine samples. Gas chromatography– mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) are the main techniques used in the majority of these methods.
3
Page 3 of 34
Sample preparation techniques used in these chromatographic methods include the conventional liquid-liquid extraction (LLE) [3, 7, 10], solid-phase extraction (SPE) [4, 9, 11-14] and the use of polyvinylidene difluoride (PVDF) filter
ip t
syringes [15]. Miniaturized techniques, such as solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) have been scarcely employed
cr
for this purpose [2, 16].
Over the last decade, the development of miniaturized extraction
us
procedures, especially LPME, has become an encouraging field in analytical
an
chemistry. In hollow fiber-liquid phase microextraction (HF-LPME), a water immiscible solvent is immobilized as a thin supported liquid membrane (SLM)
M
into the pores of a porous hollow fiber. The lumen of the fiber is filled with acceptor solution and the system is placed in contact with the sample (donor
d
phase). The analytes are extracted from the sample (aqueous), through the
te
SLM (organic) and finally into the acceptor solution. The acceptor solution can be an organic solvent, providing a two-phase extraction system or it can be an
Ac ce p
aqueous solution, providing a three-phase extraction system. In the three-phase extraction system, analytes must be ionizated, and consequently, trapped in the lumen of the hollow fiber. Due to the high sample-to-acceptor volume ratio, very high analyte enrichments can be obtained by the use of HF-LPME, especially in the three-phase mode. Also, excellent clean-up has been reported from complex biological matrices because the pores size of the fiber provides microfiltration of macromolecules [17, 18]. In the three-phase mode, besides obtaining a better recovery compared to two-phase, this technique allows easy drying and subsequent derivatization of the analytes, a chemical reaction that increases the selectivity and stability of
4
Page 4 of 34
the compounds. Therefore, an interesting perspective of three-phase mode is the possibility of performing a totally “green analytical chemistry” by the use of fatty oils or essential oils as SLM instead of using organic solvents. This type of
ip t
extraction can eliminate the use of hazardous organic solvents in sample preparation methods based on HF-LPME [18, 19].
cr
The aim of the present study was to develop a method for the
determination of KT and its main metabolites (NK and DHNK) in urine samples
us
using hollow-fiber liquid phase microextraction (HF-LPME) in three-phase mode
an
and gas chromatography-mass spectrometry (GC–MS). The method was fully validated and succesfully applied to urine samples collected from two clinical
M
cases, confirming the suspicion of ketamine exposure in these cases.
d
2. Experimental
te
2.1. Reagents and reference standards
Ac ce p
Ketamine (KT), norketamine (NK) and dehydronorketamine (DHNK)
solutions (1.0 mg/mL) in methanol and the internal standards (IS) ketamine-d4 (KT-d4) and norketamine-d4 (NK-d4) solutions (1.0 mg/mL) also in methanol were purchased from Cerilliant Analytical Reference Standards® (Round Rock, TX, USA). Dihexyl ether, 1-nonanol, undecane, decanol, 1-octanol, xilol, trifluoroacetic anhydride (TFAA) and ethyl acetate were purchased from SigmaAldrich® (MO, USA), while sodium hydroxide and hydrochloric acid were purchased from Merck® (Darmstadt, Germany). Essential oils of canola, clove and peppermint were obtained from Mapric® (São Paulo, SP, Brazil). Essential oil of eucalyptus was purchase from Mapric® (São Paulo, SP, Brazil) and
5
Page 5 of 34
Natural Pharma Ltda® (São Paulo, SP, Brazil), while soybean oil and olive oil from Bunge Brazil® (São Paulo, SP, Brazil) were obtained from a local grocery.
ip t
2.2. Preparation of standard solutions Working solutions of KT, NK, DHNK at concentrations of 1.0 µg/mL and
cr
0.1 µg/mL were prepared with methanol in volumetric glassware. Stock
solutions were stored refrigerated (-20ºC) when not in use. Working solutions of
us
the IS (KT-d4 and NK-d4) at a concentration of 1.0 µg/mL were also prepared in
an
methanol. 2.3. Instrumentation
M
Hollow-fiber Q3/2 Accurel KM polypropylene (600 µm i.d., 200 µm wall thickness and 0.2 µm pore size) was purchased from Membrana (Wuppertal,
d
Germany). Gel-loading pippeter tips Round CC 4853 (0.5 mm; 1-200 µL) were
te
purchased from Costar (Corning, NY, USA). Extractions were conducted using
Ac ce p
a multi-tube vortexer model VWR VX-2500 (Thorofare, NJ, USA). The analyses were performed using an Agilent 6850 Network GC System gas chromatograph coupled with an Agilent 5975 Series quadrupole mass seletive detector (MSD) (Wilmington, DE, USA). Samples were injected into the GC-MS by means of an autosampler (Agilent 7693). Injections were made in the splitless mode (2 min and afterwards split vent was turned on in a ratio of 1:50). Chromatographic separation was achieved on a HP-5MS fused-silica capillary column (30 m x 0.25 mm x 0.1 µm film thickness) using helium as the carrier gas at 1.0 mL/min in a constant flow rate mode. The column oven temperature program was as follows: first held at 100°C, then programmed at 10°C/min to 200°C (hold 1 min), then 5°C/min at 210°C (hold 1 min); 40°C/min at 300°C (hold 1 min). 6
Page 6 of 34
Injection port and transfer line were 260°C and 280°C respectively. The MS was operated by electron ionization (70 eV) in selected ion monitoring (SIM) mode. The following ions were chosen for SIM analyses (quantification ions
ip t
underlined): KT: 270, 262, 305; KT-d4: 274, 266; NK: 284, 256, 214; NK-d4: 288, 260; DHNK: 214, 282, 317. NK-d4 was also used as IS for DHNK.
cr
2.4. Urine samples
us
The drug-free urine samples were obtained from nonuser volunteers (laboratory staff) and were stored at –20°C until analysis. The drug-free
an
samples were used for the validation of the analytical method. The urine samples collected from real cases were obtained from the Campinas Poison
M
Control Center, Faculty of Medical Sciences, State University of Campinas, Brazil. The protocol of study was previously approved by the Faculty of
d
Pharmaceutical Sciences Ethics Committee, University of São Paulo, Brazil
te
(Ethics Protocol Approval n° CEP 98156).
Ac ce p
2.5. Sample preparation
An aliquot of 2.0 mL of urine was transferred into a 4 mL glass tube,
followed by the addition of 20 ng of each IS (KT-d4 and NK-d4). The sample was alkalinized with NaOH 2 mol/L solution until pH 10 and added 10% NaCl (m/v). A 7.5-cm hollow fiber was filled-up with eucalyptus oil in its pores. The fiber lumen was filled with 18 µL of HCl 1.0 mol/L using a gel-loading pipetter tip and the ends of the hollow-fiber were sealed by mechanical pressure with pliers. The fiber was then introduced as U-shape into the sample solution. During the extraction, the assembly was submitted for shaking at 2400 rpm for 30 minutes in the multi-tube vortexer. After extraction, the acceptor phase was 7
Page 7 of 34
withdrawn from the fiber and dried under nitrogen stream at 40ºC. The residue was derivatized with 100 µL of TFAA: ethyl acetate (1:1) at 60ºC for 30 min. After cooling, the samples were dried once more (40ºC under nitrogen stream)
ip t
and resuspended with 35 µL of ethyl acetate. An aliquot of 2.0 µL of this solution was injected into the GC–MS system.
cr
2.6. Optimization of the method
us
The study of optimization of the method was performed taking into consideration the choice of SLM, pH of donor phase, influence of acceptor
an
phase, time for extraction, intensity of shaking and addition of salt to the extraction yield. Fortified urine samples at a concentration of 15 ng/mL of each
M
analyte were submitted to the method previously described. The efficiency of extraction was evaluated by the absolute area produced by each analyte in all
d
tested conditions. The following parameters were studied: SLM (dihexyl ether,
te
xilol, 1-nonanol, decanol, 1-octanol, undecane, clove oil, eucalyptus oil,
Ac ce p
peppermint oil, canola oil, soybean oil and olive oil); pH of donor phase (pH 9, 10, 11, 12 and 13); acceptor phase (0.010, 0.025, 0.050, 0.075, 0.10 and 1.0 mol/L HCl); time for extraction (5, 10, 15, 20, 30 and 45 min); agitation (800, 1200, 1600, 2000 and 2400 rpm). The salting out effect was also tested by adding 0, 5, 10 and 20 % of NaCl (m/v) to the sample before extraction. All the optimization procedure was performed in three replicates for each parameter evaluated. The other fixed parameters set as constant while changing a given variable were those described in section 2.5 (Sample preparation). 2.7. Validation of the method
8
Page 8 of 34
The validation of the method was carried out by establishing both limit of detection (LoD) and quantitation (LoQ), selectivity/specificity, robustness, linearity, intra and inter-assay accuracy, precision, recovery, and dilution
ip t
integrity after optimization of the method. All validation procedures were performed in six replicates for each parameter evaluated. Specific topics about
cr
validation of the method are described below.
us
2.7.1. Limit of detection (LoD) and limit of quantification (LoQ)
The limit of blank (LoB) was established to ensure a reliable LoD. The
an
LoB is the highest apparent concentration expected to be found when 60 replicates of 20 different blank samples containing no analytes are tested. The
[20].
d
blank)
M
data are expressed in the equation LoB = mean blank + 1.645 (standard deviation
te
The LoD can be defined as the lowest concentration of analyte in a sample which can be reliably distinguished from the LoB and at which detection
Ac ce p
is feasible. The procedure to determinate the LoD was similar to how the LoB was determined, by using low concentrations of the analyte spiked into negative urine samples and using the equation LoD = LoB + 1.645 (standard deviation low concentration sample).
When it is used 1.645 standard deviation, no more than 5% of
the values should be less than the LoB. If the observed LoD sample values meet this criterion, the LoD is considered established or verified. The LoQ can be defined as the lowest concentration of a sample that can still be quantified with acceptable precision and accuracy. The acceptance criteria were values < 20% (RSD) for precision and accuracy between 80-120% [20, 21]. 2.7.2. Interference studies 9
Page 9 of 34
Interference studies were evaluated by assaying ten different drug-free urine samples and 10 potential interfering drug compounds. Urine samples were extracted and analyzed according to the previously described procedure
ip t
for assessment of endogenous substances. Peaks at the retention time of interest were compared to those from urine samples spiked with the analytes at
cr
the LOQ. The method was also evaluated for potential interfering substances through the analysis of blank samples fortified with 1000 ng/mL of methamphetamine,
benzoylecgonine,
11-nor-9-carboxy-
us
amphetamine,
an
tetrahydrocannabinol, diazepam, morphine, codeine, acetylsalicylic acid, naphazoline and caffeine. Acceptance criteria for this assay was the absence of
M
interfering substances at the retention times of the analyte(s) of interest and of their respective IS [6, 21].
d
2.7.3. Robustness
te
Robustness is a measure of the ability of a method to remain unaffected
Ac ce p
by small changes that might occur during analysis. In this case, possible differences in the analysis by using essential oils of eucalyptus deriving from different manufacturers were evaluated. Two sets of urine samples containing known concentrations of 1.5, 12.5 and 40.0 ng/mL of all analytes (six replicates each) were analysed according to the method described in the section 2.5 (Sample preparation). In one set, eucalyptus oil was deriving from Mapric® (São Paulo, SP, Brazil). In another set, eucalyptus oil was deriving from Natural Pharma Ltda® (São Paulo, SP, Brazil). 2.7.4. Linearity
10
Page 10 of 34
Linearity was established by analyzing urine samples containing all the analytes of interest at the following concentrations: 0.5, 1, 2.5, 5, 10, 25 and 50 ng/ml of KT, NK and DHNK. Six replicate samples were analyzed at each
ip t
concentration level.
cr
2.7.5. Precision and accuracy
The precision and accuracy study was performed by analyzing urine
us
samples containing known concentrations of 1.5, 12.5 and 40 ng/mL of KT, NK and DHNK during three consecutive days. The analyses were performed in six
an
replicates for each day. Precision, defined as the relative standard deviation (RSD), was determined by intra and inter-day repetitions. Experimental
M
concentrations were obtained using the standard calibration curves. Accuracy was expressed as a percentage of the known concentration, i.e., mean
Ac ce p
2.7.6. Recovery
te
d
measured concentration/nominal concentration × 100.
The recovery studies were performed by preparing two sets of urine
samples. One set of samples (set A), consisting of three concentrations (1.5, 12.5 and 40 ng/mL) for each analyte, were analyzed in six replicates for each concentration according to the method described in Section 2.5 (Sample preparation). The second set (set B), also comprised samples in six replicates for each concentration (1.5, 12.5 and 40 ng/mL). However, for set B, the analytes were spiked into the samples immediately after LPME procedure. Absolute recovery was evaluated by comparison of the mean response obtained for the set A (processed) and the response of set B (unprocessed). The unprocessed response represented 100% recovery. 11
Page 11 of 34
2.7.7. Dilution integrity Dilution integrity is a parameter that allows evaluating samples with levels of analytes above the calibration curve. When dilution integrity is
ip t
necessary, it should be demonstrated by spiking the matrix with analytes above the highest concentration of the curve and then diluting this sample with blank
us
should be within the set criteria, i.e. within ±15% [22].
cr
matrix (at least five determinations per dilution factor). Accuracy and precision
an
2.8. Aplication to real cases
To prove the applicability of this analytical method, real cases that
M
involved ketamine exposure were analyzed. The two cases corresponded to two women (35 and 27 years old) who misused ketamine as a suicide attempt
d
or for supposed criminal purposes. Urine samples were collected at the hospital
te
in all the cases. The quantification was based on the ratios of the ion peak areas of the compounds to the IS ion peak areas. The calibration cures were
Ac ce p
used to determine both ketamine and its metabolite concentrations.
3. Results and discussion 3.1. Sample preparation
The majority of methods developed to detect ketamine and metabolites in urine samples comprise of conventional liquid-liquid extraction (LLE) or solid phase extraction (SPE) as sample preparation techniques, before chromatographic analysis. However, these techniques require relatively large volumes of organic solvents that are toxic to the analyst and hazardous to the environment. In fact, 12
Page 12 of 34
the so-called “green chemistry” has gained considerable interest in a broad field of the analytical area, because it reduces exposure and/or waste of toxic agents from sample preparation. For this reason, miniaturized techniques, such as
ip t
SPME and LPME, have adopted this philosophy and use little or no organic solvent in their procedures [19].
cr
In the scientific literature, only two recent publications considered the
utilization of SPME or LPME for the analysis of ketamine and its metabolites in
chromatographic–mass
spectrometric
(SPME-GC-MS)
an
microextraction-gas
us
urine [2, 16]. Brown et al [2] developed and validated a solid-phase
method for the determination of four “club drugs” (gamma-hydroxybutyrate,
M
ketamine, methamphetamine and methylenedioxymethamphetamine) in human urine. However, SPME presents some disadvantages: it is expensive, the
d
fragile fiber has a limited lifetime and due to the high cost, there is the possibility
te
of sample carry-over effect because the same fiber may be used for multiple extractions. LPME, on the other hand, has eliminated the carry-over effect since
Ac ce p
the hollow fibers can be discarded after each extraction due to their low cost [18].
Xiong
et
al
[16]
developed
a
HF-LPME
combined
with
gas
chromatography-flame ionization detection (GC-FID) for the simultaneous quantification
of
amphetamine,
methamphetamine,
methylenedioxyamphetamine, methylenedioxymethamphetamine, caffeine and ketamine in drug abuser urine samples. The LoD obtained in their study for ketamine was 8.0 ng/mL. In the present work, the LoDs obtained for KT, NK and DHNK were 0.25, 0.1 and 0.1 ng/mL, respectively. Depending on the configuration used, there is a limitation related to the number of samples that can be processed in the same batch. Here we used the
13
Page 13 of 34
U-shape configuration without any supporters connecting the hollow-fiber ends. Due to the simplicity of these LPME units, many samples can be processed at the same time, providing a high sample throughput, similar to others works [23,
ip t
24]. The major disadvantage is the lack of automation of the process since it is a relatively new technique.
cr
Despite the additional step in the sample preparation and the toxicity of the TFAA, derivatization was required for the GC-MS analysis. Without the use
us
of derivatizing reagents, overlapping of the chromatographic peaks between
an
underivatized KT and DHNK was observed. Trifluoroacetic anhydride (TFAA), a low cost and commonly used acylating reagent, showed to be the best choice
M
as a derivatization agent for KT and its metabolites [14]. It allowed the detection of low levels of KT and its metabolites with an excellent chromatographic
d
separation among the analytes. Mass spectra of the derivatizated KT, NK and
te
DHNK obtained in full scan mode are shown in Fig. 2. In summary, at least 40 urine samples could be processed in less than 3
Ac ce p
hrs by one analyst alone, considering the entire procedure from sample preparation until injection into the chromatographic system. 3.2. Optimization
For the optimization of the method, different pHs of the donor phase
(sample solution) and the acceptor phase were tested. KT, NK and DHNK are basic drugs and urine samples were adjusted from pH 9 to13 using a solution of 2 mol/L NaOH. The best pH of urine sample for the extraction of these analytes was 10, but a decrease in the signal for DHNK was observed at pH values > 11, similar to the findings of Sams and Pizzo [25]. Probably this behavior occurs due to the alpha-beta unsaturated ketone present in the cyclohexenone ring of 14
Page 14 of 34
the DHNK. In this condition, NaOH acts as nucleophile which reacts in the betacarbon and generates enolate, an intermediate ion stabilizated by resonance between negative charge of the alpha-carbon and carbonyl group [26]. This
ip t
particular aspect of DHNK could have an influence on the low capacity of extraction of this compound at pH > 11. Taking into consideration the acceptor
cr
phase, a concentration of HCl greater than 0.025 mol/L slightly increased the
response. However, concentrations greater than 1.0 mol/L produced a
us
dramatically reduction of the peak area of KT and irregular responses for NK
an
and DHNK. Probably the high concentration of acid solution caused degradation of these molecules. Therefore, HCl 1.0 mol/L was chosen as
M
acceptor phase.
Different organic solvents were also tested as SLM for the LPME system.
d
The best organic solvent for the analytes was dihexyl ether. Essential and fatty
te
oils were evaluated in comparison to dihexyl ether. Dihexyl ether was the best organic solvent for extraction of KT, while NK and DHNK were better extracted
Ac ce p
using essential oil of eucalyptus (Fig. 3). Therefore, essential oil of eucalyptus was chosen to be used as SLM in the present method. By replacing the organic solvent for an essential oil, it was possible to obtain a totally “green chemistry” approach for the extration procedure in the present method. The equilibrium of analytes between donor phase and acceptor phase
can be established more rapidly with the agitation of LPME system. In this work, the effect of agitation velocity on the extraction yield of the analytes was investigated by shaking the LPME system at different agitation rates (800, 1200, 1600, 2000 and 2400 rpm) by using a multi-tube vortexer. The experimental results showed that the peak areas of KT, NK and DHNK were increased at
15
Page 15 of 34
2400 rpm. Subsequently, an agitation rate of 2400 rpm was chosen for further studies. Extraction time using HF-LPME for KT, NK and DHNK started from 5 to
ip t
45 min. Maximum peak area for the analytes were obtained with 30 min of agitation. Surprisingly, at 45 min, all analytes presented a decrease in the
cr
response. Therefore, 30 min was selected as ideal time for extraction. Moreover, salting-out effect was evaluated by increasing NaCl concentration
us
from 0 to 20% (m/v) in sample solution. The concentration of 10% of salt
an
provided higher peak area and better chromatograms. 3.3. Validation of the method
M
The validation of the method was carried out by establishing selectivity/specificity, robustness, linearity, recovery, intra and inter-day
d
precision, accuracy, limit of detection (LoD), limit of quantification (LoQ) and
te
dilution integrity. No interfering peaks due to endogenous or exogenous
Ac ce p
substances were observed at the retention time of the compounds of interest. The robustness tests results showed differences less than 20% (83.4–112.1%) were observed between samples analyzed by using eucalyptus oils from two different suppliers in three levels of concentration. Other confidence parameters of the validated method are summarized in the Table 1. After optimization of the extraction, the recovery values for the study of
KT, NK and DHNK ranged from 64.6 to 101%. These results can be considered excellent since recoveries exceeding 80–90% are rare due to analyte trapping within the SLM in the three-phase mode of LPME [18]. For quantification by HFLPME, the use of deuterated labelled IS for each analyte is recommended to improve the precision of the method. In this work, KT-d4 and NK-d4 were used 16
Page 16 of 34
as IS for their respective analogues and NK-d4 was also used as IS for DHNK, since the deuterated analogue for this substance is not commercially available. Nevertheless, precision was considered acceptable for all tested substances
ip t
(RSD < 13.5% for intra-day precision). The calibration curves were linear over the specified range (0.5–50 ng/mL). The linear regression equations and the
cr
respective relative standard deviation (RSD) were: Y= 0.1103X+0.0032, r2= 0.9996 (KT); Y= 0.1022X+0.0513, r2 = 0.9985 (NK), and Y= 0.084X+0.1149, r2=
us
0.9936 (DHNK), where Y and X represent the relationship between the peak
an
area ratio (compound/IS) and the corresponding calibration concentrations. The values of accuracy for these analytes at three different concentration levels
M
ranged from 86.2 to 95.9%.
The LoD and LoQ obtained for the analytes were below the minimum
d
required performance limit - MRPL (1.0 ng/mL for ketamine and norketamine)
te
established by UNODC [6]. This limit was set taking into consideration cases of drug-facilitated sexual assault (DFSA). DFSA incidents are generally reported
Ac ce p
later than 24h after the alleged assault, when very little drug still remains in the victim’s body. A summary of developed methods to detect ketamine and its metabolites in urine samples is shown in Table 2. As it can be observed, the LoQs obtained in the present method are lower than those reported by the references [2-4, 7, 10-16, 27-30] and higher than the ones reported in the reference [9], who worked with UPLC-MS/MS. Only a few studies consider the use of hydrolysis procedures or the detection of hydroxylated compounds for the analysis of KT and metabolites in urine samples [7, 31] (Table 2). Lin and Lua [7] analyzed 24 paired urine samples (with and without hydrolysis procedure) and they obtained the
17
Page 17 of 34
hydrolyzed and unhydrolyzed ratio for each analyte. The obtained H/U ratios were in the range of 0.85-1.85, 1.05-2.22 and 0.80-1.87 for KT, NK and DHNK, respectively. However, hydrolysis step is an additional and lengthy process, and
ip t
our method showed that hydrolytic cleavage was not necessary to achieve the recommended maximum detection limit (1.0 ng/mL) established by UNODC for
cr
the targeted analytes (KT and NK) [6, 8] and also for DHNK.
Due to the possibility of urine samples with concentrations of KT, NK and
us
DHNK above the calibration range, the study of the dilution integrity was done
an
to ensure that the method is precise and accurate after this procedure. Dilution integrity was performed by spiking the blank urine with 350 and 3500 ng/mL and
M
diluting this sample with the same matrix in 10 and 100 times (six replicates per dilution factor). Accuracy of these dilutions for KT, NK and DHNK ranged from
d
86.20 to 95.90%, while the precision (RSD) was below 10.30%. These results
te
are in accordance with criteria established by EMA (RSD + 15%) [22].
Ac ce p
3.4. Application of the method
The developed HF-LPME and GC-MS method was applied for the
quantification of KT and its metabolites in two real cases handled by the Campinas Poison Control Center (SP-Brazil). These cases are described below:
Case 1: a 35 year-old woman disappeared from her workplace for 10
days and she was found unconscious in a car. When she woke up, the woman said she had been kidnapped, but she disappeared again and she was later found unconscious in a bathroom of shopping mall. The patient was admitted to the hospital with Glasgow coma scale 10, 120/70 mmHg of blood pressure and
18
Page 18 of 34
normal respiratory rate. She remained in the intensive care unit during 24 hours and reported her suicide attempt. Case 2: a 27 year-old woman was found in a sugar-cane plantation with
ip t
burns in her body and unstable neurological status, alternating between periods of confusion and lucidity. When the patient arrived at the hospital, her blood test
cr
showed leukocytosis and hypernatremia, while other blood exams showed normal values. A urine sample was collected 2 hours after admittance in the
us
hospital. First, she was suspected of attempted rape, but the gynecologist
an
dismissed this hypothesis.The patient was then discharged from the hospital after an 8-days stay.
M
KT, NK and DHNK were detected in both urine samples from the two cases and the results are presented in the Table 3. In the case 1, where
d
concentrations were above the highest level of the calibration curve, samples
te
were diluted appropriately with blank urine to fit into the calibration curve. Relatively high levels of ketamine and its metabolites were found in the urine
Ac ce p
sample and they are in accordance with the case history (suicide attempt). In the case 2, in spite of the gynecologist dismissed the hypothesis of attempted rape, the possibility of drug-facilitated crime should not be ignored. Concentrations of ketamine and norketamine were a litlle higher than the minimum required performance limit - MRPL (1.0 ng/mL, according to the UNODC guidelines) [6]. Fig. 4 shows chromatograms obtained with the practical use of this method for the analyses of urine samples.
4. Conclusion
19
Page 19 of 34
The results showed that the HF–LPME/GC-MS method is well suited for the determination of ketamine and its main metabolites in human urine samples. Compared to other methods, it revealed some practical advantages such as
ip t
simple device, relatively low cost and sensitivity. In addition, no organic solvent was required for the extraction as an essential oil (eucalyptus oil) was used as a
cr
SLM, providing a green bioanalytical method. The method developed in this
us
study can be useful in the fields of both clinical and forensic toxicology.
an
5. References
[1] E. Visser, S.A. Schug, The role of ketamine in pain management, Biomed.
M
Pharmacother. 60 (2006) 341-348.
[2] S.D. Brown, D.J. Rhodes, B.J. Pritchard, A validated SPME-GC-MS method
te
171 (2007) 142-150.
d
for simultaneous quantification of club drugs in human urine, Forensic Sci.Int.
Ac ce p
[3] K. Lian, P. Zhang, L. Niu, D. Bi, S. Liu, L. Jiang, W. Kang, A novel derivatization approach for determination of ketamine in urine and plasma by gas chromatography-mass spectrometry, J.Chromatogr. A 16 (2012) 104109.
[4] E.M. Kim, J.S. Lee,S.K. Choi, M.A. Lim, H.S. Chung, 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), Forensic Sci. Int. 174 (2008) 197-202. [5] M.K. Shbair, S. Eljabour, M. Lhermitte, Drugs involved in drug-facilitated crimes: part I: alcohol, sedative-hypnotic drugs, gamma-hydroxybutyrate and ketamine. A review, Ann. Pharm. Fr. 68 (2010) 275-285. 20
Page 20 of 34
[6] United Nations Office on Drugs and Crime (UNODC). Guidelines for the forensic analysis of drug facilitating sexual assault and other criminal acts 2011.
United
Nations,
New
York,
2011.
Available
from:
ip t
http://www.unodc.org/documents/scientific/forensic_analys_of_drugs_facilitati ng_sexual_assault_and_other_criminal_acts.pdf. (accessed 15/05/ 2013).
cr
[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.
us
28 (2004) 181-186.
an
[8] Society of Forensic Toxicologists (SOFT). Recommended minimum performance limits for common DFSA drugs and metabolites in urine
M
samples. United States of America, Mesa, 2012. Available from: http://softtox.org/sites/default/files/SOFT_DFSA_Rec_Det_Limits_3-2012.pdf.
d
(accessed 15/05/ 2013).
te
[9] M.K. Huang, C. Liu, J.H. Li, S.D. Huang, Quantitative detection of ketamine, norketamine, and dehydronorketamine in urine using chemical derivatization
Ac ce p
followed by gas chromatography-mass spectrometry, J. Chromatogr. B. 820 (2005) 165-173.
[10] P.S. Cheng, C.Y. Fu, C.H. Lee, C. Liu, C.S. Chien, GC-MS quantification of ketamine, norketamine, and dehydronorketamine in urine specimens and comparative study using ELISA as the preliminary test methodology, J. Chromatogr. B 852 (2007) 443-449.
[11] C.Y. Chen, M.R. Lee, F.C. Cheng, G.J. Wu,Determination of ketamine and metabolites in urine by liquid chromatography-mass spectrometry, Talanta 72 (2007) 1217-1222.
21
Page 21 of 34
[12] M.C. Parkin, S.C. Turfus, N.W. Smith, J.M. Halket, R.A. Braithwaite, S.P. Elliott, M.D. Osselton, D.A. Cowan, A.T. Kicman ,Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem
ip t
mass spectrometry, J. Chromatogr. B. 876 (2008) 137-142. [13] N. Harun, R.A Anderson, E.I. Miller, Validation of an enzyme-linked
cr
immunosorbent assay screening method and a liquid chromatographytandem mass spectrometry confirmation method for the identification and
us
quantification of ketamine and norketamine in urine samples from Malaysia,
an
J. Anal. Toxicol. 33 (2009) 310-321.
[14] H.R. Lin, A.C. Lua, Simultaneous determination of amphetamines and
M
ketamines in urine by gas chromatography/mass spectrometry, Rapid Commun. Mass Spectrom. 20 (2006) 1724-1730.
d
[15] T. Nema, E.C. Chan, P.C. Ho, Extraction of ketamine from urine using a
te
miniature silica monolithic cartridge followed by quantification with liquid chromatography tandem mass spectrometry (LC-MS/MS), J. Sep. Sci. 34
Ac ce p
(2011) 1041-1046.
[16] J. Xiong, J.Chen, M.He, B. Hu, Simultaneous quantification of amphetamines, caffeine and ketamine in urine by hollow fiber liquid phase microextraction
combined
with
gas
chromatography-flame
ionization
detector, Talanta 82 (2010) 969-975.
[17] J. Lee, H.K. Lee, K.E. Rasmussen, S. Pedersen-Bjergaard, Environmental and bioanalytical applications of hollow fiber membrane liquid-phase microextraction: a review, Anal. Chim. Acta. 624 (2008) 253-268.
22
Page 22 of 34
[18] S. Pedersen-Bjergaard, K.E. Rasmussen, Liquid-phase microextraction with porous hollow fibers, a miniaturized and highly flexible format for liquidliquid extraction,J. Chromatogr. A. 1184 (2008) 132-142.
ip t
[19] S. Pedersen-Bjergaard, K.E. Rasmussen, Liquid-phase microextraction utilising plant oils as intermediate extraction medium--towards elimination of
cr
synthetic organic solvents in sample preparation, J. Sep. Sci. 27 (2004) 1511-1516.
us
[20] D.A. Armbruster, T. Pry, Limit of blank, limit of detection and limit of
an
quantitation, Clin. Biochem. Rev.29 (2008) S49-52.
[21] F.T. Peters, O.H. Drummer, F. Musshoff, Validation of new methods,
M
Forensic Sci. Int. 165 (2007) 216-224.
[22] European Medicine Agency (EMEA). Guideline on bioanalytical method
d
validation. United Kingdom, London, 2011.
Muñoz,
M.D.
te
[23] R.A. Menck, D.S.D. Lima, S.C. Seulin, V. Leyton, C.A. Pasqualucci, D.R. Osselton,
M.
Yonamine,
Hollow-fiber
liquid-phase
Ac ce p
microextraction and gas chromatography-mass spectrometry of barbiturates in whole blood samples, J. Sep. Sci. 35 (2012) 3361-3368.
[24] L. N. Pantaleao, B.A.B. Paranhos, M. Yonamine, Hollow-fiber liquid-phase microextraction of amphetamine-type stimulants in human hair samples, J. Chromatogr. A. 7 (2012) 1-7.
[25] R. Sams, P. Pizzo, Detection and identification of ketamine and its metabolites in horse urine, J. Anal. Toxicol. 11 (1987) 58-62. [26] Aldehydes and ketones: nucleophilic addition reactions, in: J. Mc Murry, Organic Chemistry – 6th edition, Cengage Learning, Stamford, 2005, pp. 704705.
23
Page 23 of 34
[27] H.H. Lee, J.F. Lee, S.Y. Lin, P.H. Chen, B.H. Chen, Simultaneous determination of HFBA-derivatized amphetamines and ketamines in urine by gas chromatography-mass spectrometry, J. Anal. Toxicol.35 (2011) 162-169.
ip t
[28] M.M.R. Fernandez, M. Laloup, M. Wood, G. De Boeck, M. Lopez-Rivadulla, P. Wallemacq, N. Samyn, Liquid chromatography-tandem mass spectrometry for
the
simultaneous
analysis
of
multiple
hallucinogens,
cr
method
chlorpheniramine, ketamine, ritalinic acid, and metabolites, in urine, J. Anal.
us
Toxicol. 31 (2007) 497-504.
an
[29] Y. Fan, Y.Q. Feng, S.L. Da, X.P. Gao, In-tube solid-phase microextraction with poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary
M
for direct high-performance liquid chromatographic determination of ketamine in urine samples, Analyst 129 (2004) 1065-1069.
d
[30] S.L. Chou, M.H. Yang, Y.C. Ling, Y.S. Giang, Gas chromatography-isotope
te
dilution mass spectrometry preceded by liquid-liquid extraction and chemical derivatization for the determination of ketamine and norketamine in urine, J.
Ac ce p
Chromatogr. B799 (2004) 37-50.
[31] R. Moaddel, S.L. Venkata, M.J. Tanga, J.E. Bupp, C.E. Green, L. Iyer, A. Furimsky, M.E. Goldberg, M.C. Torjman, I.W. Wainer, A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome, Talanta 82 (2010) 1892-1904.
Legends Figure 1. Chemical structure of target drug analytes.
24
Page 24 of 34
Figure 2. Mass spectra in full scan mode and chemical structure of TFAA derivatives of ketamine, norketamine and dehydronorketamine; A) Ketamine; B) Norketamine; C) Dehydronorketamine. Figure 3. Bar graphs showing efficiencies of ketamine, norketamine and
ip t
dehydronorketamine with the use of six different supported liquid membranes for the hollow fiber–liquid phase microextraction procedure. SLM, supported liquid membrane.
cr
Figure 4. Chromatogram obtained with the analysis of ketamine in urine using
HF-LPME. (A) blank urine sample; (B) urine sample spiked at 10.0 ng/mL of KT,
us
KT-D4, NK, NK-D4 and DHNK; (C) real urine sample containing 7.3, 5.3 and 6.8 ng/mL of KT, NK and DHNK respectivaly. KT, ketamine; KT-D4, ketamineNK,
norketamine;
NK-D4,
norketamine-deuterated;
DHNK,
an
deuterated;
Ac ce p
te
d
M
dehydronorketamine.
25
Page 25 of 34
Table 1. Confidence parameters of the validated method for the determination of ketamine, norketamine and dehydronorketamine in urine samples (six replicates for each point). Confidence parameters
KT
NK
C1
101.0
94.3
C2
91.1
88.5
C3
85.2
86.9
LoD (ng/mL)
0.25
0.10
LoQ (ng/mL)
0.50
DHNK
C2
10.1
C3
cr
68.1
us an
7.5
0.50
64.6
0.10 0.50
9.2
9.3
6.7
13.5
2.9
4.7
9.9
9.1
6.4
16.9
7.3
5.9
9.8
3.0
3.6
14.1
108.0
105.4
118.9
88.3
92.0
106.3
99.0
94.9
111.7
10 times
0.9
1.7
7.0
100 times
2.0
8.0
10.3
10 times
95.9
95.4
86.2
100 times
92.6
93.3
88.7
M
C1
69.7
d
Intra-day precision (%RSD)
ip t
Recovery (%)
Inter-day precision (%RSD)
te
C1 C2
Ac ce p
C3 Accuracy (%) C1 C2 C3
Dilution integrity
Precision (%RSD)
Accuracy (%)
26
Page 26 of 34
C1, 1.5 ng/mL; C2, 12.5 ng/mL; C3, 40 ng/mL; 10 times, 350 ng/mL; 100 times, 3500 ng/mL; LoD, limit of detection; LoQ, limit of quantification; Precision and accuracy was performed during three consecutive days; %RSD, relative standard deviation; KT,
Ac ce p
te
d
M
an
us
cr
ip t
ketamine; NK, norketamine; DHNK, dehydronorketamine.
27
Page 27 of 34
Table 2. Summary of analytical methods for the determination of KT and its
Extraction
Detection
LoQ (ng/mL)
Reference
KT
SPME
GC-MS
500
2
KT
LLE
GC-MS
10
3
KT and NK
SPE
GC-PCI/MS
50
KT and NK
LLE
GC-MS
5 and 10
7
KT and NK
SPE
UPLC-MS/MS
0.10
9
KT, NK and DHNK
LLE
GC-MS
20, 20 and 30
10
KT
SPE
LC-MS/MS
1.60
11
KT, NK and DHNK
SPE
GC-MS
25, 30 and 50
12
KT, NK and DHNK
SPE
GC-MS
1.5
13
KT, NK and DHNK
SPE
GC-MS
15, 10 and 20
14
KT, NK and DHNK
PVDF filter
LC-APCI/MS
3.17, 1.60 and 1.10
15
KT
HF-LPME
GC-FID
30
16
KT and NK
SPE
LC-MS/MS
0.50 and 20
27
KT
SPME
LC-UV
21.30
28
KT and NK
LLE
GC-MS
13 and 9
29
KT and NK
SPE
MECK
5
30
cr
us
an
d
te
ip t
Analytes
M
major metabolites in urine samples.
4
SPME, solid phase microextration; LLE, liquid-liquid extraction; SPE, solid
Ac ce p
phase extraction; PVDF, polyvinylidene difluoride; HF-LPME, hollow fiber-liquid phase microextraction; GC-MS, gas chromatography-mass spectrometry; PCI, positive chemical ionization; LC-UV, liquid chromatography-ultraviolet; FID, flame ionization detector; UPLC, ultra performance liquid chromatography; MECK, micellar electrokinetic chromatography
28
Page 28 of 34
Table 3. Results of the analysis of urine samples from real cases. Urine samples
KT (ng/mL)
NK (ng/mL)
DHNK (ng/mL)
Case 1
87.3
5805
8760
Case 2 7.3 5.3 KT, ketamine; NK, norketamine; DHNK, dehydronorketamine.
Ac ce p
te
d
M
an
us
cr
ip t
6.8
29
Page 29 of 34
Acknowledgments
Acknowledgments Financial supports from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e
Ac
ce pt
ed
M
an
us
cr
ip t
Tecnológico (CNPq) are gratefully acknowledged.
Page 30 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 1
Page 31 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure 2
Page 32 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 33 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 4
Page 34 of 34
