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Review Review: The Application of Liquid Electrochemical Detection for the Determination of Drugs of Abuse

Kevin Honeychurch

Centre for Research in Biosciences, Department of Applied Sciences, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, UK; [email protected]; Tel.: +44-117-3287357

Academic Editor: Frank L. Dorman Received: 22 June 2016; Accepted: 29 August 2016; Published: 22 September 2016

Abstract: This review (4 tables, 88 references) describes current developments in the design and application of liquid chromatography electrochemical detection (LC ED) based approaches for the determination of drugs of abuse. Specific emphasis is placed on operating details and performance characteristics for selected applications. LC ED has been shown to be highly sensitive and specific as well being a more economic option. A wide range of abused substances have been determined using this approach, including: , , opiates, , mushroom toxins, benzodiazepines and several legal highs. Reverse-phase liquid chromatography with either amperometric or coulometric determination has been the most commonly reported applications. However, coulometric arrays have been also reported. Detection limits in the ng/mL region have been reported for most target analytes.

Keywords: cannabinoids; ethanol; opiates; morphine; mushroom toxins; benzodiazepines; legal highs; liquid chromatography; electrochemical detection

1. Introduction Drugs of abuse can be defined as drugs that are taken for nonmedicinal reasons (usually for mind-altering effects); which can to physical and mental damage and with some substances, dependence and addiction. The abuse of drugs is a significant public health problem affecting almost every community and family playing a role in a wide range of social problems, such as driving violations, violence, stress, child abuse and other crimes. The misuse of drugs can also result in homelessness, and employment problems. Nevertheless, controversially, a number of these same compounds have been the subject of recent studies [1–4] that have shown their potential therapeutic properties. As a result there is a pressing need for analytical techniques capable of determining these drugs and their metabolites in different sample matrices. The application of liquid chromatography with spectrometry [5–8] and [6,8] have been widely used and have been reviewed recently. The application of for the determination of morphine has been reviewed by Tagliaro et al. [9]; however, this present review represents the first on the liquid chromatographic electrochemical detection (LC ED) for the determination of drug of abuse.

2. Principles of Operation and Practical Considerations More extensive and in-depth treatment of the fundamentals of LC ED can be found in a number of reviews and monographs [10–15]. A simple explanation of liquid chromatography with electrochemical detection would show that target analytes are separated chromatographically via their interactions with the stationary phase (column) and mobile phase. After separation, the compounds present within

Separations 2016, 3, 28; doi:10.3390/separations3040028 www.mdpi.com/journal/separations Separations 2016, 3, 28 2 of 29

Separations 2016, 3, 28 2 of 29 the mobile phase enter the electrochemical detector. A number of different electrochemical detector systemscompounds have been present utilised; within including the mobile conductivity, phase enter potentiometric, the electrochemical amperometry detector. and A coulometry. number of With amperometricdifferent electrochemical or coulometric detector based systems detectors have compounds been utilised; areincluding either conductivity, oxidized or poten reduced,tiometric, leading to amperometry and coulometry. With amperometric or coulometric based detectors compounds are the consumption (reduction) or liberation (oxidation) of at the interface. The current either oxidized or reduced, leading to the consumption (reduction) or liberation (oxidation) of formedelectrons from this at the is linearly electrode related interface. to theThe current formed of fro them analyte this is linearly and hence related can to be the used for quantification.concentration Two of the different analyte modes and hence of electrochemical can be used for quantification. detection are Two generally different employed, modes of either amperometryelectrochemical or coulometry. detection are These generally differ employed, in a number either ofamperometry ways, but or essentially coulometry. on These the geometrydiffer of the workingin a number electrode of ways, and but essentially the way, on and the how geometry much of ofthe the working analyte electrode interacts and the with way the, and electrode: how in amperometricmuch of the detection analyte analytesinteracts with flow theover electrode:the working in amperometric electrode surface detection and analytes in coulometric flow over ,the analytesworking flow electrodebetween surfaces surface and of the in working coulometric electrode electrodes, leading analytes to greater flow between conversion surfaces efficiencies. of the leading to greater conversion efficiencies. 2.1. Thin Layer Amperometric Detector 2.1. Thin Layer Cell Amperometric Detector A numberA number of different of different amperometric amperometric detectors detectors have have been been described, described, but mostbut most commonly commonly described are thedescribed wall jet are and the the wall thin jet and layer the cell. thin The layer thin cell. layer The thin cell layer (Figure cell 1(FigureA) is based 1A) is based on the on design the design originally describedoriginally by Kissingerdescribed by et al.Kissinger [16]. Theet al. design [16]. The allows design for allows a smooth for a smooth flow offlow eluent of eluent over over the the electrode surface;electrode limiting surface; baseline limiting noise baseline facilitating noise facilitating better detection better detection limits. Thelimits. small The small cell size cell alsosize a allowslso for low deadallows volumes for low dead aiding volumes in the aiding overall in chromatographicthe overall chromatographic performance. performance.

FigureFigure 1. Amperometric 1. Amperometric detector detector schematics: schematics: (A) channel; (A) channel; (B) wall-jet; (B) wall (-ajet;) entrance; (a) entrance; (b) exit; (b) (exit;c) working (c) working electrode; (d) spacer gasket; after Weber and Purdy [10]. electrode; (d) spacer gasket; after Weber and Purdy [10]. 2.2. Wall-Jet Amperometric Detector 2.2. Wall-Jet Amperometric Detector Figure 1B shows the configuration of this amperometric detector. The wall-jet configuration Figureemploys1B a showsnozzle or the jet configuration through which ofsolution this amperometric flows. The stream detector. or jet of Thethis wall-jet impinges configuration employsperpendicularly a nozzle or onto jet throughthe working which electrode. solution Fleet flows.and Little The [17 stream] reported or on jet the of thisadvantages solution of the impinges perpendicularlygeometry. The onto cell thedesign working is reported electrode. less susceptible Fleet and to Littlefowling; [17 presumably] reported ondue theto the advantages cleansing of the geometry.action Theof the cell solution design jet. isMore reported importantly, less susceptible the design is topurposed fowling; to also presumably increase the due amount to the of cleansingthe action of the solution jet. More importantly, the design is purposed to also increase the amount of the analyte arriving at the working electrode surface. However, as the flow of the jet onto the electrode surface can cause some degree of turbulence possible increases in baseline noise can result. Separations 2016, 3, 28 3 of 29 Separations 2016, 3, 28 3 of 29

analyte arriving at the working electrode surface. However, as the flow of the jet onto the electrode 2.3. Multi-Electrodesurface can cause Amperometric some degree Detectorsof turbulence possible increases in baseline noise can result. It is possible to use more than one working electrode which can be arranged in a number of 2.3. Multi-Electrode Amperometric Detectors configurations; such as in series, parallel or opposing (Figure2). Recently, the application of these multiple electrodeIt is possible detection to use more has than been one reviewed working byelectrode Honeychurch which can [ 18be]. arranged Using in such a number approaches of it configurations; such as in series, parallel or opposing (Figure 2). Recently, the application of these is possible to obtain not just quantitative analytical data on concentration but also electrochemical multiple electrode detection has been reviewed by Honeychurch [18]. Using such approaches it is confirmationpossible ofto theobtain eluting not just compound. quantitative Chao analytical et al. data [19 ] on have concentration described but the also application electrochemical of parallel modeconfirmation amperometric of the detection eluting compound. (Figure2a). Chao In et this al. mode,[19] have two described working the electrodesapplication of are parallel held atmode different potentialsamperometric points along detection the hydrodynamic(Figure 2a). In this wave mode of, two the target working analyte. electrodes The are ratio held of at these different currents can thenpotentials be measured points along and the used hydrodynamic to confirm wave the identityof the target and analyte. peak purityThe ratio of of the these eluting currents compound. can Furtherthen to be this, measured it is possible and used to useto confirm the parallel the identity detector and inpeak a different purity of the mode, eluting tomeasure compound. both Further oxidisable and reducibleto this, it is species possible simultaneously, to use the parallel by detector applying in a different mode, potential to measure to each both electrode. oxidisable Co-eluting and compoundsreducible with species different simultaneously, potentials by applying can be determined different potential by selecting to each the electrode. potential Co of- oneeluting electrode compounds with different redox potentials can be determined by selecting the potential of one so that only the more easily oxidised (or reduced) compound is detected, while at the other parallel electrode so that only the more easily oxidised (or reduced) compound is detected, while at the other electrodeparallel both electrode compounds both compounds are electrochemically are electrochemically detected. detected. The concentration The concentration of the secondof the second compound can hencecompound be calculated can hence by be difference. calculated by difference.

Figure 2. Parallel and series configurations for amperometric dual electrode detection systems. Figure 2. Parallel and series configurations for amperometric dual electrode detection systems. W1 = working electrode 1; W2 = working electrode 2. (a) Parallel; (b) Series and (c) Parallel adjacent. W1 = workingArrow indicates electrode direction 1; W of2 = flow. working electrode 2. (a) Parallel; (b) Series and (c) Parallel adjacent. Arrow indicates direction of flow. Figure 2b shows the configuration of the amperometric series mode. This configuration uses the Figuretwo working2b shows electrodes the configuration in the flow channel of the independently amperometric potentiostatically series mode. This controlled. configuration This can usesbe the two workingcompared electrodes to fluorescence in the detecti flowon channel with the independently product of the upstream potentiostatically electrode being controlled. detected Thisat the can be downstream working electrode. When using amperometric cells such as the thin-layer cell (TLC) in compared to fluorescence detection with the product of the upstream electrode being detected at the the series mode only a small percentage of the compounds passing through the downstream downstream working electrode. When using amperometric cells such as the thin-layer cell (TLC) in the series mode only a small percentage of the compounds passing through the downstream generator cell will be electrochemically oxidised or reduced. The same is true for the second upstream amperometric electrode, which will in turn convert only a fraction of the products generated by the first electrode. Nevertheless, such an approach has been shown to be successful for the determination of a number of benzodiazepines [20,21] and nitro aromatic compounds [22]. Separations 2016, 3, 28 4 of 29

The related parallel adjacent approach has been used by Evans [23] for the determination of several organic peroxides. The configuration of this detector allows for the cascade of reversible redox reactions in order to amplify the detector current. In this configuration, the working electrodes are on opposite sides of a very thin channel, with one electrode at an oxidative potential and one at a reducing potential. When the spacer is thick, then the two electrodes can act as independent parallel dual electrodes, but with a thin spacer products of one electrode can diffuse to other and vice versa. When the electrodes are sufficiently large, each analyte particle can pass through a number of oxidation-reduction cycles, so that the conversion efficiency of the cell can be much larger.

2.4. Pulse Amperometric Detection Pulsed integrated amperometry (PIA) uses a multistep potential-time waveform that rapidly alternates between an amperometric detection mode with oxidative cleaning reductive reactivation potentials. It provides significant enhancement of sensitivity for compounds generally considered as non-electroactive for detection under constant applied potential and with poor optical properties for spectrophotometric detection (carbohydrates, amines, thiols) and macromolecules of biological importance (, ). PIA is based on the capacity of Au to catalyse the oxidation of organic compounds [24]. The unsaturated d- orbitals present in the Au can bind and stabilise free-radical intermediates generated during electrochemical oxidation, promoting from the oxidised analyte to the Au surface [25] hence allowing for the quantification of the target analyte. This approach can lead to the accumulation of oxidation products at the surface and the eventually fouling and leading to the loss sensitivity. To overcome this, potential is step to a positive potential for a short limited time to desorb the surface contaminants via the formation of Au oxides [26]. The potential is then stepped to a third, negative value to reduce the Au oxides formed, regenerating the original clean Au surface. Such multi-step waveforms are repeated continuously through the duration of the analysis. For measurement of amino acids and amino sugars, a different series of steps is used to maintain slightly higher potentials during detection such that the Au surface is maintained in an oxide state [25]. For the determination of saccharides, liquid chromatography with-PIA detection is reported to be two orders of magnitude more sensitive than LC with refractive index detection [27].

2.5. Coulometric Detectors A number of different coulometric detector designs have been developed, with the majority of reported applications utilizing the commercially available Coulochem detector. A simplified cross section of this is shown in Figure3. These utilise flow-through porous carbon electrodes with high surface areas reducing diffusion distances giving close to 100% conversion efficiency (coulometric). These high conversion rates of the analyte passing through them as predicated by Faraday’s Law can result in high sensitivity. However, the larger currents generated do not necessarily lead to improvements in signal-to-noise ratios or to detection limits due to the concomitant increase in noise. Nevertheless, these high coulometric efficiencies offer a number of other potential advantages when two or more of these electrodes are used in the series after the liquid chromatographic column. When two or more electrodes are arranged in series these can be applied in what is commonly referred to as the screening mode. Compounds eluting from the analytical column entry the first upstream electrode cell which is held at a potential high enough to oxidise or reduce, and hence remove some of the possible interfering compounds. By careful selection of the potential of this first screening electrode, the target analytes can pass through unaltered, and can be measured at the downstream detector electrode, but now in the absence of a number of the potential interferences originally present in the sample. A variation of this concept has led to the development of detectors consisting of up to sixteen coulometric detectors in series, to form a coulometric array; the application of which has been extensively reviewed [28–31]. A further alternative method is what has been termed the generator/detector mode. Here the first upstream working electrode can be used as a generator, SeparationsSeparations2016, 20163, 28, 3, 28 5 of 295 of 29

extensively reviewed [28–31]. A further alternative method is what has been termed the withgenerator/detector the second working mode. electrode,Here the first as theupstreamdetector working. The generatorelectrode can electrode be used canas a be generator used to, with form an electrochemicalthe second working active product, electrode, which as the candetector then. The be measured generator electrode at the second can be downstream used to form detector an electrodeelectrochemical giving the active advantage product, of which it being can much then be more measured easily at oxidized the second or reduceddownstream than detector the parent compound.electrode Other giving possible the advantage interferences of it being present much in more the sampleeasily oxidized extract willor reduced be irreversibly than the parent reduced or compound. Other possible interferences present in the sample extract will be irreversibly reduced or oxidized by the downstream generator electrode, similar to the screening mode, and will not be seen oxidized by the downstream generator electrode, similar to the screening mode, and will not be seen at the upstream detector. Due to the lower potentials now required at the detector a number of other at the upstream detector. Due to the lower potentials now required at the detector a number of other advantagesadvantages are gained,are gained, such such lower lower background background currentscurrents and and less less potential potential interferences. interferences.

Figure 3. Cross section through a dual coulometric electrode showing the placement of working, Figure 3. Cross section through a dual coulometric electrode showing the placement of working, counter and palladium- reference electrodes, after Honeychurch [18]. counter and palladium-hydrogen reference electrodes, after Honeychurch [18]. 3. Applications 3. Applications 3.1. Cannabinoids 3.1. Cannabinoids A number of different chromatographic approaches have been employed for the determination Aof numbercannabis ofand different its constituent chromatographic cannabinoids approaches and have been have reviewed been employed [32]. HPLC for-UV the procedures determination are of cannabiswidely and used its for constituent the analyses cannabinoids of the cannabinoids and have utilising been reviewedwavelengths [32 between]. HPLC-UV 215 and procedures 280 nm. are widelyHowever, used for the thesensitivity analyses of the of electrochemical the cannabinoids detector utilising has been wavelengths shown to be between over 400 times 215 and greater 280 nm. However,than that the obtained sensitivity by UV of at the 220 electrochemicalnm [33]. Around 70 detector different has been shown compounds to be are over reported 400 times present in herbal [34], however, Δ9- (I) (THC) is the major greater than that obtained by UV at 220 nm [33]. Around 70 different cannabinoid compounds psychoactive component and methods for its determination9 are consequently more commonly are reportedreported. presentNyoni et in al. herbal [35] have cannabis developed [34], a however, LC ED based∆ -Tetrahydrocannabinol method to assist in investigating (I) (THC) the is the majorbiochemical psychoactive mechanism(s) component of andTHC methods(I) in brain for tissue. its determination The method employed are consequently extraction more commonly with reported.methanol Nyoni- et- al.ethyl [35 acetate,] have followed developed by analysis a LC ED using based LC ED. method Overall to recoveries assist in were investigating reported the biochemicalto be greater mechanism(s) than 80%. of THC (I) in brain tissue. The method employed solvent extraction with methanol-hexane-ethylBourquin and Brenneisen acetate, followed [36] utilised by analysis reverse phase using LC LC ED ED. for Overall the determination recoveries wereof the reportedmajor to be greatermetabolite than of 80%. THC, 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (II) (THC–COOH) in urine. BourquinSeparation andwas achieved Brenneisen using [36 a] mobile utilised phase reverse of methanol phase- LC5% EDaqueous for the acetic determination acid (76:24) at of a flow the major- metaboliterate of 1.5 of mL/min. THC, 11-nor- The detector∆9-tetrahydrocannabinol-9-carboxylic was operated in the amperometric mode, acid at (II) an (THC–COOH) applied potential in of urine. +1.2 V (vs. Ag/AgCl). A 10 mL aliquot of human urine was fortified with Separation was achieved using a mobile phase of methanol-5% aqueous acetic acid (76:24) at a (cannabinol) and hydrolysed with KOH by heating to convert the excreted glucuronide conjugates flow-rateto free, of unconjugated 1.5 mL/min. THC The– detectorCOOH (II). was The operated sample inpH the was amperometric then adjusted mode,to pH 5 at–6 anand applied the internal potential of +1.2standard V (vs. and Ag/AgCl). THC–COOH A (II) 10 isolated mL aliquot by solid of humanphase extraction urine was (SPE) fortified [37]. Both with THC internal–COOH standard (II) (cannabinol)and the internal and hydrolysed standard were with reported KOH by to have heating the same to convert recovery the of excreted 90% ± 5%. glucuronide The limit of detection conjugates to free,for unconjugated THC–COOH THC–COOH (II) was 5 ng/mL (II). ofThe urine sample based pH on was a signal then-to adjusted-noise-ratio to pH of 5: 5–61. The and standard the internal standardcalibration and THC–COOH curve was obtained (II) isolated by using by blank solid urine phase spiked extraction with 25 (SPE)–300 [ng/mL37]. Both THC THC–COOH–COOH (II) and (II) and the internal90 ng/mL standard internal werestandard. reported to have the same recovery of 90% ± 5%. The limit of detection for THC–COOH (II) was 5 ng/mL of urine based on a signal-to-noise-ratio of 5:1. The standard was obtained by using blank urine spiked with 25–300 ng/mL THC–COOH (II) and 90 ng/mL internal standard. Separations 2016, 3, 28 6 of 29

Separations 2016, 3, 28 6 of 29

(I)

(II)

(III)

(IV)

(V)

Separations 2016, 3, 28 7 of 29

Separations 2016, 3, 28 7 of 29

(VI)

Further investigations [38] into the simultaneous determination of THC and the further metabolites THC–COOHTHC–COOH and 11-hydroxy-11-hydroxy-Δ∆9--tetrahydrocannabinoltetrahydrocannabinol (III) (III) (11 (11-OH-OH THC) THC) in urine and plasma by LC ED have been reported. Biological fluids fluids were firstfirst hydrolysed by heating in base and then acidifiedacidified and extracted with a previously described automatic extractor [ 39] and determined by LC ED with amperometric determination at +1.1 V. V. Liquid chromatographychromatography was carried out with a reversed-phase silica C8 column and a mobile phase of acetonitrile/methanol/0.02 N H2SO4 (35:15:50) reversed-phase silica C8 column and a mobile phase of acetonitrile/methanol/0.02 N H2SO4 (35:15:50) at a flow flow rate of 1.8 mL/min. mL/min. A A detection limit of of under under 0.5 0.5 ng/mL ng/mL (S/N (S/N > 3) > with 3) with a linear a linear range range of 10 of– 10–500500 ng/mL ng/mL was was reported reported.. Using Using this this approach approach it itwas was found found possible possible to to detect detect THC THC–COOH–COOH (II) in rabbit urine up to 216216 hh afterafter administration.administration. Investigations were also made on samples of human urine obtained from 23 men arrested on the suspicion of smoking marijuana and results sh showedowed good agreement with those obtained by GC/MSGC/MS [ [4040].]. THC has also been determined in human urine using a novel brominated 9 9-carboxy-11–nor--carboxy-11–nor-Δ∆9-- tetrahydrocannabinol as as an an internal internal standard standard [41 [41].]. Amperometric Amperometric determination determination was was undertaken undertaken at at+0.85 +0.85 V V (vs (vs.. Ag/ Ag/AgCl)AgCl) using using gradient gradient . elution. Problems Problems generally generally exist exist with with the the application of gradient elution whenwhen usedused inin conjugationconjugation with with LC LC ED. ED. Electrochemical Electrochemical detector detector background background current current is ais function a function of organic of organic solvent solvent composition composition and ionic and strength.ionic strength. Consequently, Consequently, the application the application of solvent of gradientsolvent gradient will result will in result a continually in a continually changing changing background background current. current.However, However, the authors the showed authors thatshowed this that can this be minimized can be minimized by making by making the ionic the strengths ionic strengths of the two of the components two components of the mobileof the mobile phase identical.phase identical. Using Using this approach this approach the authors the authors reported reported only aonly small a small shift shift in the in baseline the baseline of around of around 1 to 21 nA.to 2 AfternA. After addition addition of internal of internal standard standard samples samples of human of human urine wereurine adjusted were adjusted to be between to be between pH 4.5 andpH 4.5 pH and 6.5 andpH 6.5 isolated and isolated by SPE. by No SPE. interferences No interferences were reported were reported for 22 drugs for and22 drugs metabolites. and metabolit A pooledes. relativeA pooled standard relative deviationstandard deviation of 4.1% (n of= 4.1 27)% was (n = obtained27) was obtained for the quality for the control quality samplescontrol samples and the methodand the showed method good showed agreement good with agreement results withobtained results by gas obtained chromatography/mass by gas chromatography/. spectrometry.Nakahara and Tanaka [42] have develop a chemotaxonomical based LC ED method for the discriminationNakahara of and confiscated Tanaka [42 cannabis] have develop samples. aEleven chemotaxonomical different samples based were LC investigated ED method andfor the on thediscrimination basis of their of liquid confiscated chromatographic cannabis samples. cannabinoid Eleven profiles. different They samples demonstrated were investigated the possibility and on of distinguishingthe basis of their cannabis liquid chromatographic products grown cannabinoid in three districts profiles. of Japan, They Colombia,demonstrated The the Philippines possibility and of Nepal.distinguishin The stabilityg cannabis of cannabinoids products grown in cannabis in three productsdistricts of was Japan, also examinedColombia, atThe room Philippines , and 4Nepal.◦C and The−20 stability◦C. After of cannabinoids 16 months, it wasin cannabis found that products 90% of was original also THCexamined (I) remained at room at temperature,−20 ◦C, 50% and4 °C 28%and of−20 original °C. After THC 16 (I)months, had decomposed it was found at that room 90% temperature of original andTHC 4 (◦I)C, remained respectively. at −20 Almost °C, 50 all% ofand the 28 decomposed% of original THCTHC (I)(I) hadhad changeddecomposed to cannabinol at room temperature by air oxidation. and 4 °C, respectively. Almost all of theNakahara decomposed and THC Sekine (I) [had42] changed have used to LCcannabinol ED for theby air determination oxidation. of free cannabinoids and cannabinoicNakahara acids and obtained Sekine from [42] marijuana have used cigarettes LC ED for and the in tar determinatio and ash obtainedn of free by using cannabinoids an automatic and smokingcannabinoic . acids Separation obtained from was achieved marijuana using cigarettes reverse and phase in chromatographytar and ash obtained with mobile by using phase an automatic smoking machine. Separation was achieved using reverse phase chromatography with of 0.02 N H2SO4, methanol acetonitrile (6:7:16) at a flow rate of 1.1 mL/min. Amperometric detection wasmobile undertaken phase of using 0.02 anN Happlied2SO4,-methanol potential aceton of +1.2itrile V(vs. (6:7:16) Ag/AgCl) at a flow and arate liner of range 1.1 mL/min. of 5 to 500Amperometric ng/injection detection was reported was undertaken with detection using limits an applied of 0.5 to potential 0.9 ng/injection of +1.2 V for (vs free. Ag/AgCl) cannabinoids and a andliner 1.2 range to 2.5 of ng/injection5 to 500 ng/injection for cannabinoic was reported acids with (S/N detection > 4). THC limits (I) and of 0.5 several to 0.9 related ng/injection cannabinoids: for free cannabinoids and 1.2 to 2.5 ng/injection for cannabinoic acids (S/N > 4). THC (I) and several related cannabinoids: cannabidiol (IV), cannabinol (V), cannabichromene (VI) and their acid derivatives could be resolved using the chromatographic conditions described. In its pure synthetic form THC has been developed as the drug dronabinol, which has been used for its anti-emetic and orexigenic effects in cancer patients receiving chemotherapy. Kokubun et al.

Separations 2016, 3, 28 8 of 29 cannabidiol (IV), cannabinol (V), cannabichromene (VI) and their acid derivatives could be resolved using the chromatographic conditions described. In its pure synthetic form THC has been developed as the drug dronabinol, which has been used for its anti-emetic and orexigenic effects in cancer patients receiving chemotherapy. Kokubun et al. [43] have developed a LC ED amperometric method to investigate the pharmacokinetics of dronabinol in cancer patients and its quantitation in blood. Reverse phase chromatography was undertaken using and a mobile phase of 50 mM KH2PO4/CH3CN (9:16). Detection was undertaken using an applied potential of +0.40 V. The calibration curve was linear in the range of 10 ng/mL to 100 ng/mL. The lower limit of quantification was 0.5 ng/mL (S/N = 3). The relative within-runs and between-runs standard deviations for the assay were less than 4.7%. Table1 gives a summary of the LC ED approaches reported for the determination of cannabinoid compounds.

Table 1. Liquid chromatography electrochemical determination of cannabinoids.

Reference LC ED Technique Linear Range Detection Limit Comments Ref. Electrode Amperometric mode; 5 ng/mL of urine Ag/AgCl 25–300 ng/mL THC–COOH in urine [36,37] +1.2 V (S/N = 5) Amperometric mode; ∆9-tetrahydrocannablnol Ag/AgCl Up to 10 µg/mL 1.5 ng on column [35] +1.2 V levels in brain tissue THC and metabolites; Amperometric mode; Ag/AgCl 10–500 ng/mL 0.5 ng/mL (S/N > 3) THC–COOH) and 11-OH THC [38] +1.1 V in rabbit and human urine THC–COOH is Brominated 9-Carboxy-11-nor Amperometric mode; 0.012 µg/mL Ag/AgCl 0.012–0.20 µg/mL ∆9 tetrahydrocannabinol as [41] +0.85 V (limit of quantification) internal Standard in human urine Chemotaxonomical discrimination of confiscated cannabis. Studies made on Amperometric mode; stablity of cannabinoids in Ag/AgCl 1–500 µg/mL – [42] +1.0 V herbal cannabis. Mobile phase: CH3CN/CH3OH/0.02 N H2SO4. Benozic acid as internal standard Limit of Amperometric mode; Blood THC levels in patients Ag/AgCl 10–100 ng/mL quantification: [43] +0.40 V given the drug dronabinol 0.5 ng/mL (S/N = 3) 0.5 to 0.9 ng/injection for free cannabinoids Cannabinoic contents in Amperometric mode; and 1.2 to Ag/AgCl 5–500 ng/injection marijuana cigarettes and in tar [33] +1.2 V 2.5 ng/injection for and ash cannabinoic acids (S/N > 4).

3.2. Ethanol The determination of the degree of alcohol consumption is an important parameter commonly studied in forensic investigations. In post-mortem investigations, the presence of ethanol can be a result of either consumption prior to death, or due to fermentation as part of the decomposition process. To be able to differentiate between the two possibilities, a common approach is to monitor the biological metabolites for ethanol generated before death, such as ethyl glucuronide (VII) (EtG). However, alkyl-glucuronides such as EtG only adsorb at low UV wavelengths, and hence have little analytical utility. In light of this a method for the determination of EtG has been recently developed [44] using reversed-phase liquid chromatography coupled with pulsed electrochemical detection (PED) [45]. EtG was quantified using methyl glucuronide as an internal standard, and was separated using a mobile phase consisting of 1% acetic acid/acetonitrile (98/2, v/v). Post-column addition of NaOH (600 mM) at 0.5 mL/min allowed for the detection of all glucuronides using PED at a gold working electrode vs. Ag/AgCl. A limit of detection of 0.03 µg/mL for a 50 µL injection volume was reported Separations 2016, 3, 28 9 of 29 with a coefficient of variation of 1.7% at the limit of quantitation. Sample clean-up and isolation was achieved via SPE using an aminopropyl phase cartridge, gaining a recovery of approximately Separations 2016, 3, 28 9 of 29 50% ± 2%. Investigation of 29 post-mortem urine specimens were undertaken, and results were found to agreedisolation strongly was achieved with certified via SPE determinations. using an aminopropyl phase cartridge, gaining a recovery of approximately 50% ± 2%. Investigation of 29 post-mortem urine specimens were undertaken, and results were found to agreed strongly with certified determinations.

(VII)

3.3. 3.3. Alkaloids Previously, Tagliaro et al. [9] have reviewed the LC ED applications for the determination Previously, Tagliaro et al. [9] have reviewed the LC ED applications for the determination of of morphine. Schwartz and David [46] have explored the liquid chromatography electrochemical morphine. Schwartz and David [46] have explored the liquid chromatography electrochemical determinationdetermination of of morphine morphine (VIII),(VIII), heroin (IX), (IX), codeine codeine (X), (X), thebaine thebaine (XI), (XI), narcotine narcotine (XII), (XII),papaverine papaverine (XIII)(XIII) and and cocaine cocaine (XIV). (XIV). Compounds Compounds were were determined determined by byreverse reverse phase phase chromatography chromatography using an using an acetonitrileacetonitrile phosphate phosphate or or acetate acetate buffer based based mobile mobile phase phase with with amperometric amperometric detection detection undertaken undertaken at +1.2at +1.2 V. TheV. The mechanism mechanism for for electrochemical electrochemical oxidation of of amines amines is isknown known to involve to involve the thelone lone pair pair of electronsof electrons on the on nitrogen the nitrogen atom [47– [5147]–51 the] the pH pH consequently consequently should should be be high high enoughenough to maintain maintain these compoundsthese compounds in their in basic their form. basic In form. aqueous In aqueous solutions this would this would require require a pH a 10pH or 10 above. or above. However, underHowever, the chromatographic under the chromatographic conditions employed conditions the employ authorsed the were authors able to were gain able good to sensitivitygain good using sensitivity using lower pH values in the 6–8 range. The authors concluded that in the presence of the lower pH values in the 6–8 range. The authors concluded that in the presence of the mobile phase mobile phase organic modifier the tertiary nitrogen atom remains in its basic un-protonated form at organic modifier the tertiary nitrogen atom remains in its basic un-protonated form at pH values lower pH values lower than that when strictly aqueous media are used. This enhancement of basicity was thanconcluded that when to strictlyresult from aqueous the organic media solvent are used. present This in enhancement the mobile phase. of basicity Limits wasof detection concluded were to result fromdetermined the organic to be solvent 0.3 ng presentfor morphine, in the 1 mobile ng for heroin, phase. and Limits 2 ng of for detection cocaine. were determined to be 0.3 ng for morphine,Sawyer 1et ng al. for[52] heroin,have developed and 2 ng a method for cocaine. for the determination of, morphine (VIII), heroin (IX) andSawyer hydromorphone et al. [52] have(XV) from developed post-mortem a method tissues. for Post the- determinationmortem samples of, of morphinewhole blood, (VIII), urine, heroin or (IX) andvitreous hydromorphone humour were (XV) assayed from post-mortem without pre-treatment. tissues. Post-mortemIt was reported samples necessary of whole to preserved blood, urine, or vitreoussamples with humour buffered were assayed fluoride/sodium without pre-treatment. azide to prevent It changes was reported in the levels necessary of heroin to preserved(IX) samplesand morphine with buffered (VIII). sodiumOne-hundred fluoride/sodium µL sample aliquots azide towere prevent taken changesand the internal in the levels standard, of heroin nalorphine, and ammonium /ammonium hydroxide buffer added. These were then extracted (IX) and morphine (VIII). One-hundred µL sample aliquots were taken and the internal standard, with 5 mL of dichloromethane:isopropanol (96:4 v/v). Following the organic phase was nalorphine, and ammonium chloride/ammonium hydroxide buffer added. These were then extracted extracted with pH 3 phosphate-citrate buffer and the solvent discarded. The resulting aqueous layer withw 5as mL then of extracted dichloromethane:isopropanol with pH 8.50 buffer and dichloromethane:isopropanol (96:4 v/v). Following centrifugation (96:4 v/v). theThe organicorganic was phase was extractedthen taken with and pH 3evaporated phosphate-citrate to dryness buffer under and nitrogen the solvent and reconstituted discarded. The in mobile resulting phase aqueous and layer wasexamined then extracted by LC with ED. pHA single 8.50 buffer-step extraction and dichloromethane:isopropanol procedure was also developed (96:4 v using/v). The a 99.5: organic0.5 was thendichloromethane taken and evaporated isopropanol to dryness solution. under However, nitrogen due to and interferences reconstituted from in endogenous mobile phase metabolites and examined by LCin urine ED. or A single-stepother commonly extraction encountered procedure drugs was not also recommended. developed using a 99.5:0.5 dichloromethane isopropanolHydrodynamic solution. However, due was to employed interferences to explore from endogenous the electrochemical metabolites behaviour in urine of the or other commonlythree target encountered analytes under drugs reverse was notphase recommended. chromatographic conditions using a mobile phase of 32% methanol, 68% 148 mM pH 7.30 phosphate buffer at a flow rate of 1.0 mL/min. An applied potential was employed to explore the electrochemical behaviour of the three of +0.5 V was reported to be the optimum applied potential for use with a Bioanalytical Systems, Inc target analytes under reverse phase chromatographic conditions using a mobile phase of 32% methanol, (BAS, West Lafayette, IN, USA) LC-3 single cell amperometric detector. Linear responses were 68%recorded 148 mM from pH 10 7.30 to 500 phosphate ng/mL morphine buffer at (V aIII), flow 62 rate to 1000 of1.0 ng/mL mL/min. hydromorphone An applied (XV), potential and 250 ofto +0.5 V was2000 reported ng/mL tofor be heroin the optimum(IX). Limits applied of detection potential for extracted for use sample with were a Bioanalytical 0.5 ng/mL morphine Systems, (VIII), Inc. (BAS, West3.1 Lafayette, ng/mL hydromorphone IN, USA) LC-3 single (XV), cell and amperometric 12.5 ng/mL heroin detector. (IX). Linear Average responses extraction were recovery recorded from 10 topercentages 500 ng/mL wer morphinee 70% morphine (VIII), 62 (VIII), to 1000 57% ng/mL hydromorphone hydromorphone (XV), 55% (XV), heroin and (IX), 250 toand 2000 78% ng/mL

Separations 2016, 3, 28 10 of 29 for heroin (IX). Limits of detection for extracted sample were 0.5 ng/mL morphine (VIII), 3.1 ng/mL hydromorphone (XV), and 12.5 ng/mL heroin (IX). Average extraction recovery percentages were 70% morphine (VIII), 57% hydromorphone (XV), 55% heroin (IX), and 78% nalorphine (XVI). Forty-five other Separations 2016, 3, 28 10 of 29 drugs were investigated as possible interferences. Out of theses only acetaminophen, aminopyrine, cyanide,nalorphine disopyramide, (XVI). Forty ketamine-five other and drugs nalorphine were investigated were found as possible to give interferences. chromatographic Out of peaks, theses but wereonly reported acetaminophen, not to interfere. aminopyrine, Material cyanide, from six disopyramide, human post-mortem ketamine casesand nalorphine of suspected were heroin found related to deathgive were chromatographic examined and peaks, the but were reported of heroin not to (IX) interfere. and morphine Material (VIII) from determinedsix human post in both- bloodmortem and urine cases showedof suspected good heroin agreement related withdeath those were obtainedexamined byand radioimmunoassay. the concentrations of heroin (IX) andZaromb morphine et al. (VIII) [53] have determined investigated in both the blood determination and urine showed of airborne good cocaine agreement (XIV) with and those heroin (IX)obtained using high-throughput by radioimmunoassay. liquid pre-concentration. Air was sampled at a rate over the range 550–700Zaromb L/min et al. [53 with] have apreferred investigated ranged the determination of 620–680 L/min. of airborne Detection cocaine limits(XIV) and of ca. heroin 1:10 13(IX)(v /v) of theusing drugs high were-throughput achieved. liquid LC adsorption ED was undertaken pre-concentration. using Air a mobile was sampled phase at of a rate over the phosphate range 550–700 L/min with a preferred ranged of 620–680 L/min. Detection limits of ca. 1:1013 (v/v) of the buffer (pH 7–7.4, 0.02 M)-acetonitrile (40:60, v/v) at a flow rate of 1.0 mL/min, at C stationary phase. drugs were achieved. LC ED was undertaken using a mobile phase of potassium phosphate18 buffer Amperometric detection was undertaken at a glassy carbon electrode at an applied potential of +1.0 V (pH 7–7.4, 0.02 M)-acetonitrile (40:60, v/v) at a flow rate of 1.0 mL/min, at C18 stationary phase. (vs.Amperometric Ag/AgCl). detection was undertaken at a glassy carbon electrode at an applied potential of +1.0 V (vs. Ag/AgCl).

(VIII) (IX)

(X) (XI)

(XII) (XIII)

(XIV) (XV)

Separations 2016, 3, 28 11 of 29

Separations 2016, 3, 28 11 of 29

Separations 2016, 3, 28 11 of 29

(XVI)

Buprenorphine (XVII), naloxonenaloxone (XVIII)(XVIII) (Suboxone(Suboxone(XVI) ®® ) and methadone (XIX) are commonly used for thethe treatmenttreatment ofof opioidopioid addiction.addiction. Somaini et al. [54] havehave reportedreported aa LCLC EDED coulometriccoulometric basedbased methodBuprenorphine for their determination (XVII), naloxone in human (XVIII) blood (Suboxone plasma utilising® ) and methadone levosulpiride (XIX) as are an commonly internal standard. used Chromatographicfor the treatment separation separationof opioid addiction. was was achieved achieved Somaini using using et aal. amobile mobile[54] have phase phase reported of of40:60, 40:60, a LC v/v vED /acetonitrilev coulometricacetonitrile-2.5-2.5 basedmM mM pH pH6.4method phosphate 6.4 phosphate for their buffer bufferdetermination at a atflow a flow rate in ratehumanof 0.6 of mL/min 0.6blood mL/min plasma with witha utilising cyano a cyano 250 levosulpiride mm 250 × mm 3.0 × mm,as3.0 an 5internal mm, µm column. 5 µ standard.m column. Dual DualelectrodeChromatographic electrode detection detection wasseparation undertaken was was undertaken achieved with the withusing conditioning thea mobile conditioning phase cell set of at40:60, cell +0.05 set v/ vV; at acetonitrile the +0.05 upstream V;- the2.5 mM upstreamscreening pH screeningcell6.4 set phosphate at − cell0.20 set V buffer atand− 0.20 theat a detector Vflow and rate the electrodeof detector 0.6 mL/min at electrode +0.60 with V. a atcyanoA +0.60rapid 250 V.clean mm A rapid- ×up 3.0 procedure clean-upmm, 5 µm procedureof column. the biological Dual of the electrode detection was undertaken with the conditioning cell set at +0.05 V; the upstream screening biologicalsamples using samples a microextraction using a microextraction by packed by packed sorbent sorbent technique technique was alsowas also reported, reported, employ employinging C8 cell set at −0.20 V and the detector electrode at +0.60 V. A rapid clean-up procedure of the biological Csorbent8 sorbent inserted inserted into into a syringe a syringe needle. needle. Calibration Calibration curves curves were were reported reported to be to belinear linear over over a range a range of samples using a microextraction by packed sorbent technique was also reported, employing C8 of0.25 0.25–20.0–20.0 ng/mL ng/mL for for buprenorphine buprenorphine (XVII) (XVII) and and norbuprenorphine norbuprenorphine (XX), (XX), 3.03.0–1000.0–1000.0 ng/mL ng/mL for sorbent inserted into a syringe needle. Calibration curves were reported to be linear over a range of methadone (XIX) and and 0.13–10.0 0.13–10.0 ng/mL ng/mL for naloxone with detectiondetection limits of of 0.08 0.08 ng/mL ng/mL for both 0.25–20.0 ng/mL for buprenorphine (XVII) and norbuprenorphine (XX), 3.0–1000.0 ng/mL for buprenorphine (XVII) and norbuprenorphine (XX), 0.9 ng/mL for methadone (XIX) and 0.04 ng/mL buprenorphinemethadone (XIX) (XVII) and and 0.13 norbuprenorphine–10.0 ng/mL for naloxone (XX), 0.9 with ng/mL dete forction methadone limits of 0.08(XIX) ng/mL and 0.04 for ng/mL both for naloxone (XVIII). The method was successfully applied to plasma samples obtained from former forbuprenorphine naloxone (XVIII). (XVII) The and method norbuprenorphine was successfully (XX), applied0.9 ng/mL to plasmafor methadone samples (XIX) obtained and 0.04 from ng/mL former heroin addicts treated with opioid replacement therapy. heroinfor naloxone addicts treated(XVIII). withThe method opioid replacementwas successfully therapy. applied to plasma samples obtained from former heroin addicts treated with opioid replacement therapy.

(XVII) (XVIII) (XVII) (XVIII)

(XIX)(XIX) (XX)(XX)

DentalDental cottons cottons and and cigarette cigarette filters filters areare often used toto filterfilter dissolved dissolved or or “cooked” “cooked” drug drug solutions. solutions. TheThe solution solution is isdrawn drawn into into the the syringe syringe throughthrough the improvisedimprovised filter filter prior prior to to injection. injection. Since Since drug drug filteringfiltering with with these these cottons cottons is is prevalent prevalent andand partpart of thethe drugdrug paraphernalia paraphernalia used used by by intravenous intravenous drug drug

Separations 2016, 3, 28 12 of 29

Dental cottons and cigarette filters are often used to filter dissolved or “cooked” drug solutions. The solution is drawn into the syringe through the improvised filter prior to injection. Since drug

Separationsfiltering with2016, 3 these, 28 cottons is prevalent and part of the drug paraphernalia used by intravenous12 drug of 29 users (IDUs) in their injection ritual, they represent an ideal source for obtaining samples for analysis. usersHuettl (IDUs) et al. havein their reported injection a high ritual, performance they represent liquid an chromatography ideal source for obtaining electrochemical samples array for detectionanalysis. Huettl(HPLC-EA) et al. [55 have] method reported for the a determination high performance of heroin liquid (IX), chromatography morphine (VIII), codeine electrochemical (X) and cocaine array detection(XIV) contained (HPLC- inEA) these [55] drug method cottons. for the Two determination different extraction of heroin methods (IX), morphine were investigated, (VIII), codeine using (X) andeither cocaine or (XIV) ethyl acetatecontained to extract in these the drugs drug from cottons. the cottonTwo different filters. Following extraction a 10 methods min incubated were investigated,at room temperature using either with water the selected or ethyl solvent,acetate to the extract samples the drugs were centrifugedfrom the cotton and filters. the supernatant Following ataken 10 min and incubated either blown at room down temperature to dryness with in the the case selected of ethyl solvent, acetate the or samples in the case were of water,centrifuged diluted and in themobile supernatant phase and taken filtered and beforeeither examinationblown down byto dryness HPLC-EA. in the Due ca tose theof ethyl heterogeneous acetate or in nature the case of the of water,drug cotton diluted samples in mobile standard phase addition and was filtered employed before to verify examination the retention by time HPLC of- theEA. target Due analytes to the heterogeneousheroin (IX), morphine nature (VIII),of the codeine drug cotton (X) and samples cocaine standard (XIV). Separation addition was and employed analysis of to the verify extracts the wereretention carried time out of using the target a CoulArray analytes gradient heroin HPLC (IX), morphine coupled to (VIII), a twelve codeine coulometric (X) and electrochemical cocaine (XIV). Separationarray detector and arranged analysis inof series the extracts after the were analytical carried column. out using The a potentialsCoulArray of gradient each of the HPLC electrodes coupled in tothe a arraytwelve was coulometric set at increasing electrochemical potentials array of (1) detector 300 mV; arranged (2) 350 mV; in (3)series 400 after mV; (4)the 450 analytical mV; (5) colu 500mn. mV; The(6) 550 potentials mV; (7) of 600 each mV; of (8) the 650 electrodes mV; (9) 700in the mV; array (10) was 750 mV;set at (11) increasing 850 mV andpotentials (12) 950 of mV.(1) 300 Sample mV, (2)separation 350 mV, was (3) 400 accomplished mV, (4) 450 using mV, an(5) end-capped500 mV, (6) 550 cyano mV, (100 (7) mm600 ×mV,4.6 (8) mm, 650 5 mV,µm, (9) CPS-2 700 Hypersil)mV, (10) 750column mV, (Thermo(11) 850 Scientific,mV and (12) Waltham, 950 mV. MA, Sample USA) sepa withration gradient was elution.accomplished Initial conditionsusing an end of- 95%capped (A) cyano30 mM (100 dibasic mm sodium× 4.6 mm, phosphate, 5 µm, CPS pH-2 2.95, Hypersil) 5% (B) column 30 mM ( dibasicThermo sodium Scientific, phosphate, Waltham, 30% MA, ethanol, USA) withpH 2.95 gradient at a flow elution. rateof Initial 1 mL/min conditions for 5 of min. 95% The (A) concentration 30 mM dibasic of sodium component phosphate, (B) was pH then 2.95, ramped 5% (B) to 3080% mM over dibasic a period sodium of 30 phosphate, min. Detection 30% limits ethanol, of the pH HPLC-EA 2.95 at a flow system rate were of 1 reported mL/min as:for 45 pg/ minµ.L The for concentrationmorphine (VIII), of component 24 pg/µL for (B) codeine was then (X), ramped 444 pg/ toµ 80L,% heroin over a (IX) period and of 576 30 pg/ minµ.L Detection for cocaine limits (XIV). of theThe HPLC authors-EA reported system were that itreported would beas: possible 4 pg/µL byfor using morphine a slightly (VIII), modified 24 pg/µL method for cod toeine determine (X), 444 pg/µL,lysergic heroin acid diethylamide (IX) and 576 (XXI) pg/µL (LSD) for cocaine and the THC(XIV). metabolites: The authors 11-hydroxy- reported that∆9-tetrahydrocannabinol it would be possible by(11-OH- using∆ a9 -THC)slightly and modified 11-nor- ∆method9-tetrahydrocannabinol-9-carboxylic to determine lysergic acid diethylamide acid (11-nor- (XXI)∆9-THC-9-COOH). (LSD) and the THCInvestigations metabolites: were also 11 made-hydroxy into-Δ the9-tetrahydrocannab possibility to associateinol (11a particular-OH-Δ9-THC) chromatographic and 11-nor profile-Δ9- withtetrahydrocannabinol different cuts of injectable-9-carboxylic drugs. acid Drug (11 cottons-nor-Δ9 were-THC obtained-9-COOH). from Investigations different areas were of Denver, also made USA intoto investigate the possibility this possibility to associate using a particular the output chromatographic of channel 12 (950 profile mV) ofwith the different HPLC-EA. cuts The of possibilityinjectable drugs.of developing Drug cottons a database were basedobtained on thesefrom different results was areas reported. of Denver, USA to investigate this possibility using the output of channel 12 (950 mV) of the HPLC-EA. The possibility of developing a database based on these results was reported.

(XXI) Using liquid chromatography dual electrode detection (LC-DED) in conjunction with UV detectionUsing Ary liquid and Róna chromatography [56] have reported dual electrodeon the determination detection (LC-DED) of morphine in conjunction(VIII) (7,8-didehydro with UV- 4,5detection-epoxy- Ary17-methylmorphinan and Róna [56] have-3,6 reported-diol) and on its the glucuronides; determination morphine of morphine-3-glucuronide, (VIII) (7,8-didehydro- morphine- 64,5-epoxy-17-methylmorphinan-3,6-diol)-glucuronide. Morphine (VIII) and its glucuronides and its glucuronides; were extracted morphine-3-glucuronide, from human plasma using morphine- Bond- 6-glucuronide. Morphine (VIII) and its glucuronides were extracted from human plasma using Elut C18 (1 mL) SPE cartridges. Average extraction efficiencies of morphine (VIII), morphine-3- glucronitideBond-Elut C 18and(1 mL)morphine SPE cartridges.-6-glucronitide Average were extraction 95.4%, 96.1 efficiencies% and 96.8 of morphine%, respectively. (VIII), morphine-3-Separations wereglucronitide achieved and using morphine-6-glucronitide a Supelcosi LC-8DB reverse were phase 95.4%, column 96.1% and(Sigma 96.8%,-Aldrich respectively., St. Louis, Separations MO, USA) were achieved using a Supelcosi LC-8DB reverse phase column (Sigma-Aldrich, St. Louis, MO, USA) with a mobile phase of 0.1 M KH2PO4 (pH 2.5)-acetonitrile-methanol (94:5:1 v/v/v), containing 4 mM pentasulphonic acid as the mobile phase. The coulometric detection system was reported to give a detection limit of 0.5 ng/mL for morphine twenty times lower than that obtainable by UV at 201 nm under the same LC conditions. The limit of quantitation of morphine by LC-DED was reported to be 1 ng/mL, compared to 10 ng/mL by UV detection. However, UV detection proved to be superior for the detection of the glucuronide metabolites. The response for morphine by LC-DED was found to

Separations 2016, 3, 28 13 of 29

with a mobile phase of 0.1 M KH2PO4 (pH 2.5)-acetonitrile-methanol (94:5:1 v/v/v), containing 4 mM pentasulphonic acid as the mobile phase. The coulometric detection system was reported to give a detection limit of 0.5 ng/mL for morphine twenty times lower than that obtainable by UV at 201 nm under the same LC conditions. The limit of quantitation of morphine by LC-DED was reported to be 1 ng/mL, compared to 10 ng/mL by UV detection. However, UV detection proved to be superior for the detection of the glucuronide metabolites. The response for morphine by LC-DED was found to linear over the range 1–30 ng/mL with morphine-3-glucronitide from 50 to 2000 ng/mL and morphine-6-glucronitide 15–1000 ng/mL. Rashid et al. [57] have developed an immuno-solid-phase extraction method followed by reverse-phase LC ED for the determination of morphine in urine. The polyclonal solid-phase extraction columns were fabricated from aldehyde-activated silica modified with polyclonal raised in sheep in response to an N-succinyl-normorphine-bovine serum albumin conjugate. Urine was diluted ten times with phosphate-buffered saline, pH 7.4 (PBS), loaded onto these solid-phase immunoextraction columns and washed with PBS and before then being eluted with 40% ethanol in PBS, pH 4. The eluted fraction was then analysed by LC ED using a cyanopropyl analytical column with a mobile phase of 25% acetonitrile in phosphate buffer–sodium lauryl sulphate at pH 2.4 with a flow rate of 1 mL/min. Electrochemical detection of morphine was performed with a Coulochem ESA model 5100A set at a potential of +0.45 V. Calibration curves were reported to be linear from 100 ng/mL to 500 ng/mL in urine. The inter-assay relative standard deviation was 10% with a corresponding recovery of 98%. Minimal binding with other opiate metabolites such as codeine (X), normorphine, norcodeine, morphine-3-glucuronide and morphine-6-glucuronide was reported. Xu et al. [58] have modified a glassy carbon electrode (GCE) with cobalt hexacyanoferrate for the liquid chromatographic amperometric determination of morphine (VIII) in rat brain microdialysates. The electrode was modified electrochemically by voltammetric cycling from 0.0 V to +1.0 V at 100 mV/s in a solution containing 0.5 mM K3Fe(CN)6 and 1.0 mM CoCl2 in 0.5 M KCl. The electrochemical properties of the cobalt hexacyanoferrate film modified GCE were investigated by using 0.1 M phosphate buffered saline (pH 4.5) as the supporting . At the unmodified GCE no voltammetric responses were recorded. At the cobalt hexacyanoferrate modified GCE a number of voltammetric responses were recorded, concluded to arise from the redox behaviour of Fe2+/Fe3+ couple. However, in the presence of 2.0 × 10−4 M morphine (VIII) at the cobalt hexacyanoferrate modified GCE a further large oxidation peak was observed. The voltammetric peak potential for this response was reported to have move to a more negative potentials compared to the direct oxidation of morphine at the unmodified bare GCE. This was concluded to result from the electrocatalytic behaviour of the cobalt hexacyanoferrate. LC ED investigations also showed that the peak current magnitude at the modified cobalt hexacyanoferrate GCE was larger than that recorded at the bare GCE, 66 nA compared to 15 nA for a 2.5 × 10−5 M morphine (VIII) standard. Liquid chromatographic separation was undertaken using a mobile phase of 10:90 (v/v) methanol-0.1 M phosphate-buffered saline (PBS, −4 pH 4.5) which contained 1.0 × 10 M Na2EDTA at a flow rate of 1.0 mL/min with an injection volume was 25 µL. The stationary phase was a HP Hypersil column (5 mm, 200 × 4.6 mm) (Thermo Scientific, Waltham, MA, USA). Amperometric detection of morphine (VIII) was performed at a potential of +0.60 V (vs. Ag/AgCl). A linear response for morphine was reported over the concentration range 1.0 × 10−6 M to 5.0 × 10−4 M (R2 > 0.990) with a corresponding detection limit of 5.0 × 10−7 M (S/N of 3). The morphine (VIII) concentration in the brain samples collected from anesthetized male (Sprague-Dawley) rats (250–300 g) sampled at different time intervals after morphine (VIII) was administered intravenously (25 mg/kg). The relative recoveries of morphine (VIII) at the perfusion rates of 12.0, 9.0, 6.0, 3.6, 3.0, 2.4, 1.5 mL/min were investigated and a perfusion rate of 3.6 mL/min was considered optimum. Morphine (VIII) could be readily detected in brain dialysate and no interferences were recorded at or around the retention time of morphine (VIII) (4.60 min). A number of studies have utilised LC ED for the investigation of the pharmacokinetics of morphine in dogs [59–61]. Most recently Aragon et al. [61] have investigated the pharmacokinetics of Separations 2016, 3, 28 14 of 29 a human oral morphine formulations consisting of both immediate and extended release components in adult Labrador Retrievers dogs. In their randomized design, 14 dogs were administered either 1 or 2 mg/kg morphine orally. Plasma samples were collected up to 24 h after drug administration and extracted by SPE. Concentrations of morphine (VIII) were determined by reverse-phase LC ED with gradient elution. The mobile phase consisted of 95% 0.01 M acetate buffer with 0.1% triethylamine and 5% acetonitrile with the pH adjusted to 4.5 with glacial acetic acid. The mobile phase gradient elutionSeparations program 2016, 3, 28 consisted of 100% of this mobile phase mixture for 8 min, 85% mobile phase14 with of 29 15% acetonitrile for 8–11 min and 100% mobile phase for 11–14 min. Separation was achieved with a elution program consisted of 100% of this mobile phase mixture for 8 min, 85% mobile phase with 4.6 × 150 mm and 5 µm particle size column maintained at 40 ◦C. Dual electrode coulometric detection 15% acetonitrile for 8–11 min and 100% mobile phase for 11–14 min. Separation was achieved with a was undertaken using a guard cell +750 mV; electrode 1 +300 mV; electrode 2 +450 mV, with electrode 2 4.6 × 150 mm and 5 µm particle size column maintained at 40 °C. Dual electrode coulometric detection used for quantification. The retention times for morphine and morpine-6-glucronide were 7.5 and was undertaken using a guard cell +750 mV; electrode 1 +300 mV; electrode 2 +450 mV, with electrode 5.5 min with limits of quantification of 7.8 ng/mL and 4 ng/mL respectively. Table2 summaries 2 used for quantification. The retention times for morphine and morpine-6-glucronide were 7.5 and reported LC ED application for the determination of alkaloids. 5.5 min with limits of quantification of 7.8 ng/mL and 4 ng/mL respectively. Table 2 summaries Fisher et al. [62] have compared the serum concentrations of morphine after administration of a reported LC ED application for the determination of alkaloids. buccal tablet (25 mg) with those obtained after intramuscular injection (10 mg). Buccal morphine was Fisher et al. [62] have compared the serum concentrations of morphine after administration of a administered to eleven healthy volunteers and intramuscular morphine was given to five preoperative buccal tablet (25 mg) with those obtained after intramuscular injection (10 mg). Buccal morphine was surgical patients. Serum morphine concentrations were assayed by LC DED in samples taken up to administered to eleven healthy volunteers and intramuscular morphine was given to five 8 h after drug administration. Electrochemical detection was carried out using a 5100A Coulochem preoperative surgical patients. Serum morphine concentrations were assayed by LC DED in samples detector fitted with a 5100 detector cell (ESA). The potential of electrode 1 was maintained at +0.25 V taken up to 8 h after drug administration. Electrochemical detection was carried out using a 5100A and electrode 2 at +0.40 V. The lower limit of detection was reported to be 0.8 ng/mL morphine Coulochem detector fitted with a 5100 detector cell (ESA). The potential of electrode 1 was maintained base. Morphine (VIII) analysis was carried out using the extraction and chromatographic procedure at +0.25 V and electrode 2 at +0.40 V. The lower limit of detection was reported to be 0.8 ng/mL described by Todd et al. [63]. Mean maximum morphine concentrations were eight times lower morphine base. Morphine (VIII) analysis was carried out using the extraction and chromatographic after buccal administration than after intramuscular injection and occurred at a mean of 4 h later. procedure described by Todd et al. [63]. Mean maximum morphine concentrations were eight times Individual morphine concentration-time profiles showed marked inter individual variability after lower after buccal administration than after intramuscular injection and occurred at a mean of 4 h administration of the buccal tablet, consistent with considerable variation in tablet persistence time on later. Individual morphine concentration-time profiles showed marked inter individual variability the buccal mucosa. after administration of the buccal tablet, consistent with considerable variation in tablet persistence Jordan and Hart [64] have investigated the determination of morphine (VIII) by liquid time on the buccal mucosa. chromatography with amperometric determination at a GCE. Using cyclic voltammetry they showed Jordan and Hart [64] have investigated the determination of morphine (VIII) by liquid that pH 11.0 was optimum for the electrochemical determination of morphine (VIII). Further chromatography with amperometric determination at a GCE. Using cyclic voltammetry they showed investigations showed that a mobile phase of 50 mM pH 11.0 phosphate buffer of containing 20% v/v that pH 11.0 was optimum for the electrochemical determination of morphine (VIII). of acetonitrile was found optimum. Hydrodynamic voltammetric investigations over the range +0.15 Further investigations showed that a mobile phase of 50 mM pH 11.0 phosphate buffer of containing to +1.10 V identified three distinct waves can be seen at +0.45 V, +0.8 V and a third at +1.0 V. Using an 20% v/v of acetonitrile was found optimum. Hydrodynamic voltammetric investigations over the applied potential of +0.45 V with hydromorphone (XXII) as an internal standard, a linear response was range +0.15 to +1.10 V identified three distinct waves can be seen at +0.45 V, +0.8 V and a third at observed from 1.2 × 10−12 to 4 × 10−10 M of morphine injected. At the applied potential of +0.45 V +1.0 V. Using an applied potential of +0.45 V with hydromorphone (XXII) as an internal standard, a (vs. Ag/AgCl) and for a signal-to-noise ratio of 3:1, the detection limit was found to be 1.24 × 10−13 M linear response was observed from 1.2 × 10−12 to 4 × 10−10 M of morphine injected. At the applied of morphine injected. potential of +0.45 V (vs. Ag/AgCl) and for a signal-to-noise ratio of 3:1, the detection limit was found to be 1.24 × 10−13 M of morphine injected.

Separations 2016, 3, 28 15 of 29

Table 2. Liquid chromatography electrochemical determination of alkaloids.

Analyte LC ED Technique Reference Electrode Linear Range Detection Limit Comments Ref. Amperometric mode; Airbourne concentrations of Cocaine and heroin Ag/AgCl 25–300 ng/mL ca. 1:1013 (v/v) [53] +1.0 V cocaine and heroin l0 to 500 ng/mL (morphine), For extracted sample was 0.5 ng/mL Heroin, morphine and Amperometric mode; 62 to 1000 ng/mL (morphine), 3.1 ng/mL Post-mortem samples of whole Ag/AgCl [52] hydromorphone +0.5 V (hydromorphone), and 250 to (hydromorphone), and 12.5 ng/ mL blood, urine, or vitreous humor 2000 ng/mL (heroin) (heroin) Morphine, heroin, Morphine base, 0.42–1.7 nM; codeine, thebaine, Amperometric mode; heroin hydrochloride, 0.3 ng for morphine, 1 ng for heroin, Comparisum with LC UV Ag/AgCl [46] narcotine, papaverine +1.2 V 1.6–6.5 nM; cocaine and 2 ng for cocaine detection made and cocaine hydrochloride, 3.1–12 nM. 4 pg/mL for morphine, 24 pg/mL Heroin, morphine, 12 channel PdH – for codeine, 444 pg/mL for heroin Analysis of drug cottens [55] codeine and cocaine electrochemical array 2 and 576 pg/mL for cocaine Morphine, LC coulometric DED in Morphine and its glucuronides morphine-3-glucuronide conjunction with UV PdH Morphine; 1–30 ng/mL Morphine; 0.5 ng/mL extracted from human plasma [56] and 2 detection by SPE morphine-6-glucuronide buprenorphine and Buprenorphine, 0.08 ng/mL for both buprenorphine norbuprenorphine, Plasma smaples from heroin norbuprenorphine, DED, screening −0.2 V and norbuprenorphine, 0.9 ng/mL PdH 3.0–1000.0 ng/mL for addicts. Levosulpiride as an [54] naloxone and detector +0.6 V 2 for methadone and 0.04 ng/mL for methadone and internal standard methadone naloxone 0.13–10.0 ng/mL for naloxone. Amperometric mode; Morphine Ag/AgCl 1.0 × 10−6 M to 5.0 × 10−4 M 5.0 × 10−7 M (S/N = 3). Rat brain dialysates. [58] +0.60 V. 25 µL injection Buccal and intramuscular Morphine LC coulometric DED PdH2 – 0.8 ng/mL morphine adminstered to [62] humans Amperometric mode; hydromorphone as an internal Morphine Ag/AgCl 1.2 × 10−12 to 4 × 10−10 M 1.24 × 10−13 M [64] +0.45 V standard Limits of quantification: Guard cell +750 mV; cell 1 Morphine LC coulometric DED PdH – [60] 2 25 ng/L morphine +300 mV; cell 2 +450 mV Limit of quantification for morphine; Morphine, Guard cell +750 mV; cell 1 LC coulometric DED PdH – 7 ng/mL and morpine-6-glucronide; [59] morpine-6-glucronide 2 +300 mV; cell 2 +450 mV 4 ng/mL Separations 2016, 3, 28 16 of 29

Table 2. Cont.

Analyte LC ED Technique Reference Electrode Linear Range Detection Limit Comments Ref. limits of quantification: morphine; Morphine, LC coulometric DED PdH – 7.8 ng/mL; morpine-6-glucronide [61] morpine-6-glucronide 2 ng/mL Detection of morphine in toad Amperometric mode; (Bufo marinus), rabbit and rat Morphine Ag/AgCl – – [65] +0.75 V skin, bovine adrenal, cerebellum, cerebel cortex Amperometric mode; Pooled human +0.650 V using plasma and plasma obtained 5-hydroxyindole or Ag/AgCl 25–300 ng/mL limit of quantitation; 10 ng/mL [66] from seven volunteers bufotenine as an (self-experimenting ) internal standard Psilocin and Limits of quantification of Column-switching. Analysis of 4-hydroxyindole-3-acetic LC coulometric DED PdH – 0.8 ng/mL and 5.0 ng/mL for [67] 2 human plasma acid psilocin and 4HIAA Separations 2016, 3, 28 17 of 29 Separations 2016, 3, 28 17 of 29

TheThe hallucinogen hallucinogen psilocybin psilocybin (XXIII) (XXIII) is isone one of the of main the main psychoactive psychoactive compounds compounds found in foundPsilocybe in Psilocybemushrooms. mushrooms. Psilocin Psilocin (XXIV) is(XXIV) readily is readily transformed transformed to the phenolic to the phenolic compound compound psilocybin psilocybin in the gutin theas a gut result as of a dephosphorylation result of dephosphorylation by alkaline by phosphatase alkaline phosphatase and is thus the and substance is thus the responsible substance for responsiblethe reported for psychedelic the reported effects. psychedelic Both effects. psilocybin Both (XXIII) psilocybin and (XXIII) psilocin and (XXIV) psilocin are (XXIV) listed asare Class listed A as(United Class A Kingdom) (United orKingdom) Schedule or I (US)Schedule drugs I under (US) drugs the United under Nations the United 1971 ConventionNations 1971 on Convention Psychotropic onSubstances. Psychotropic Interestingly, Substances. more Interestingly, recently, more psilocin recently, (XXIV) psilocin has been (XXIV) shown has to been have shown some to promise have someas a promise therapeutic as a agenttherapeutic in the agent treatment in the of trea conditionstment of conditions cluster headaches cluster headaches [3]. Lindenblatt [3]. Lindenblatt et al. [66 ] ethave al. [66 used] have LC used ED toLC determine ED to determine levels oflevels psilocin of psilocin (XXIV) (XXIV) in plasma in plasma samples samples obtained obtained from from both bothpooled pooled human human blood bankblood plasma bank and plasma plasma and obtained plasma from obtained seven from volunteers seven (self-experimenting volunteers (self- experimentingphysicians) taking physicians) oral doses taking of 0.2 oral mg doses psilocybin of 0.2 per mg kg psilocybin body weight per (maximumkg body weight 15 mg (maximum per person) 15 in mga placebo-controlled per person) in a placebo drug trial.-controlled Both liquid-liquid drug trial. andBoth automated liquid-liquid SPE and procedures automated were SPE explored procedures for the wereisolation explored of psilocin for the (XXIV). isolation The of determination psilocin (XXIV). of psilocin The determination (XXIV) obtained of frompsilocin liquid-liquid (XXIV) obtained extracted fromsamples liquid was-liquid undertaken extracted using samples reverse was phase undertaken chromatography using reverse with aphase 250 × chromatography4.0 mm C18 column with and a 250a mobile × 4.0 mm phase C18 ofcolumn 0.1 M sodiumand a mobile acetate, phase 0.1 M,of 0.1 citric M acid,sodium 0.03 acetate, mM Na 0.12EDTA M, citric pH acid, 4.1–acetonitrile 0.03 mM Na(83:172EDTAv/ vpH, 0.7 4.1 mL/min)–acetonitrile with (83:17 electrochemical v/v, 0.7 mL/min) detection with electrochemical at +0.650 V using detection 5-hydroxyindole at +0.650 V using (XXV) 5as-hydroxyindole an internal standard. (XXV) as SPE an internal extracts standard. were determined SPE extracts using were again, determined reverse phase using chromatography again, reverse phasebut, withchromatography a mobile phase but, ofwith 150 a mMmobile pH phase 2.3 potassium of 150 mM dihydrogen pH 2.3 potassium phosphate dihydrogen buffer–acetonitrile phosphate buffer(94.5:5.5–acetonitrilev/v, 0.6 mL/min) (94.5:5.5 withv/v, 160 0.6 mM mL/min) Na2EDTA with in 160 the mMbuffer–acetonitrile Na2EDTA in mixture. the buffer The–acetonitrile potential of mixture.the electrochemical The potential detector of the waselectrochemical set at +0.675 detector V with quantification was set at +0.675 carry V out with using quantification bufotenine (XXVI)carry outas anusing internal bufotenine standard. (XXVI) The as limit an internal of quantitation standard. for The both limit methods of quantitation was 10 ng/mL for both psilocin methods (XXIV). was 10However, ng/mL psilocin on-line SPE (XXIV). showed However, better recoveries on-line SPE and showed selectivity better and recoveriesreportedly andrequired selectivity less manual and reportedlyeffort and required smaller plasma less manual volumes effort of and 400 µsmallerL, compared plasma to volumes 2 mL for of liquid-liquid 400 µL, compared extraction. to 2 mL for liquid-liquid extraction.

(XXIII) (XXV)

(XXIV) (XXVI)

AA liquid liquid chromatographic chromatographic procedure procedure based basedon column on- column-switchingswitching with electrochemical with electrochemical detection hasdetection been developed has been for developed the determination for the of determination psilocin (XXIV) of and psilocin the metabolite (XXIV) and4-hydroxyindole the metabolite-3- acetic4-hydroxyindole-3-acetic acid (XXVII) (4HIAA) acid in (XXVII)human plasma (4HIAA) [67 in]. humanPlasma plasma was extracted [67]. Plasma from wasblood extracted samples from by centrifugation.blood samples Ascorbic by centrifugation. acid was then Ascorbic added acidto protect was then the addedphenol tocompounds protect the against phenol degradation compounds andagainst the degradationsamples freeze and-dried. the samples The resulting freeze-dried. residues The wereresulting then residues re-constituted were then in re-constituted water and the in pswaterilocin and and the 4HIAA psilocin (XXVII) and 4HIAA extracted (XXVII) by extractedmicrodialysis by microdialysis (mean recovery: (mean psilocin recovery: 15.1 psilocin% ± 0.85 15.1%%; 4HIAA± 0.85%; 11.0 4HIAA% ± 1.10 11.0%%). ± 1.10%).

Separations 2016, 3, 28 18 of 29

Separations 2016, 3, 28 18 of 29

Separations 2016, 3, 28 18 of 29 In order toIn avoid order excessto avoid interfering excess interfering plasma plasma compounds compounds such such as as ascorbic ascorbic acid acid reaching reaching the the detector cell a column-switchingdetector cell a column step-switching was employed. step was employed. This wasThis was achieved achieved by by connecting the theoutlet outlet of of the the injection valve was connected to a 5 cm Spherisorb RP-8 HPLC column allowing for pre- injection valveseparation was of connected the injected tosample a 5 cmdialysate. Spherisorb The outlet RP-8 of this HPLC pre-column column was allowing connected to for the pre-separation inlet of the injectedof a second sample six- dialysate.port Rheodyne The valve; outlet the flow of this of the pre-column eluate could be was directed connected either to to the the waste inlet or to of a second six-port Rheodynethe 15 cm Spherisorb valve; theRP-8 flowHPLC ofanalytical the eluate column. could The mobile be directed phase consisted either of to47% the (v/v waste) water or to the 15 cm Spherisorbcontaining RP-8 0.3 M HPLCammonium analytical acetate buffered column. to pH The 8.3 mobileby addition phase of ammoni consisteda solution of 25 47%% and (v/ v) water 53% (v/v) of methanol with a flow rate of 450 µL/min. Limits of quantification of 0.8 ng/mL and containing5.0 0.3 ng/mL M ammonium for psilocin (XXIV) acetate and 4HIAA buffered (XXVII) to pHwere 8.3 reported by addition respectively. of ammoniaThe authors noted solution that 25% and 53% (v/v)optimised of methanol liquid withchromatography a flow rate UV ofdetect 450ionµ L/min.of psilocin Limits(XXIV) resulted of quantification in a limit of quantitation of 0.8 ng/mL and of approximately 10 ng/mL; allowing for measurement of only peak plasma concentrations. Similarly, 5.0 ng/mL forIn psilocin order to (XXIV) avoid excess and 4HIAA interfering (XXVII) plasma were compounds reported such respectively. as ascorbic acid The reaching authors thenoted that GC/MS was reported to show insufficient sensitivity with the additional disadvantage of requiring a optimiseddetector liquid chromatographycell a column-switching UV step detection was employed. of psilocin This was (XXIV) achieved resulted by connecting in a limit the ofoutlet quantitation of of derivatising step. approximatelythe injection 10 ng/mL; valve was allowing connected for tomeasurement a 5 cm Spherisorb of only RP-8 peak HPLC plasma column concentrations. allowing for pre- Similarly, separation of the injected sample dialysate. The outlet of this pre-column was connected to the inlet GC/MS was3.4. reportedBenzodiazepines to show insufficient sensitivity with the additional disadvantage of requiring a of a second six-port Rheodyne valve; the flow of the eluate could be directed either to the waste or to derivatisingthe step.15The cm LC Spherisorb ED determination RP-8 HPLC of benzodiazepinesanalytical column. has The recently mobile been phase review consisted [68] and of 47has% been (v/v) shown water containingto be a highly 0.3 sensitive M ammonium and selective acetate approach. buffered Ato number pH 8.3 ofby further addition investigations of have solution been 25reported% and 3.4. Benzodiazepines53since% (thisv/v) review of methanol in 2014. with By using a flow an rate in series of 450-liquid µL/min. chromatography Limits of quantification dual electrode of detection 0.8 ng/mL in and the 5.0redox ng/mL mode, for itpsilocin is possible (XXIV) to electrochemicallyand 4HIAA (XXVII) reduce were aromatic reported nitro respectively. substituted The benzodiazepines authors noted that to The LCoptimisedtheir ED corresponding determination liquid chromatography hydroxylamine of benzodiazepines UV at thedetect firstion “generator” of haspsilocin recently electrode (XXIV)been resulted (Equation review in (a1 )limit). [68 of] andquantitation has been shown to be a highlyof approximately sensitive and 10 ng/mL; selective allowing approach. for measurement A number of only of furtherpeak plasma investigations concentrations. have Similarly, been reported Ar–NO2 + 4e− + 4H+ → Ar–NHOH + H2O (1) since this reviewGC/MS was in 2014.reported By to using show insufficient an in series-liquid sensitivity with chromatography the additional disadvantage dual electrode of requiring detection a in the derivatisingThis species step. can then be readily measured at the subsequent downstream “detector” electrode redox mode,via oxidation it is possible to the tonitroso electrochemically (Equation (2)). reduce aromatic nitro substituted benzodiazepines to their corresponding hydroxylamine at the first “generator” electrode (Equation (1)). 3.4. Benzodiazepines Ar–NHOH → Ar–N=O + 2e− + 2H+ (2)

TheThis LC is attractive ED determination analytically; of benzodiazepines as this− latter species+ has recently can be measured been review at potentials[68] and has close been to shownthat of Ar–NO2 + 4e + 4H → Ar–NHOH + H2O (1) to0 V,be a away highly from sensitive many and possible selective interfering approach. A compounds. number of furtherThis approach investigations has beenhave been used reported for the sincedetermination this review of innitro 2014. aromatic By using drug, an in nitrazepam series-liquid (XXVIII) chromatography in bovine and dual human electrode serum detection [20]. in the This speciesredox mode, can it then is possible be readily to electrochemically measured at reduce the subsequent aromatic nitro downstream substituted benzodiazepines “detector” electrode to via oxidation totheir the corresponding nitroso (Equation hydroxylamine (2)). at the first “generator” electrode (Equation (1)).

Ar–NO2 + 4e− + 4H+ → Ar–NHOH + H2O (1) → + − + + This species can thenAr–NHOH be readily measuredAr–N=O at the subsequent2e downstream2H “detector” electrode (2) via oxidation to the nitroso (Equation (2)). This is attractive analytically; as this latter species can be measured at potentials close to that of 0 V, Ar–NHOH → Ar–N=O + 2e− + 2H+ (2) away from many possible interfering compounds. This approach has been used for the determination This is attractive analytically; as this latter species can be measured at potentials close to that of of nitro aromatic drug, nitrazepam (XXVIII) in bovine and human serum [20]. 0 V, away from many possible interfering compounds. This approach has been used for the determination of nitro aromatic drug, nitrazepam (XXVIII) in bovine and human serum [20]. (XXVIII) (XXIX)

(XXVIII) (XXIX)

Separations 2016, 3, 28 19 of 29

Recently,Separations 2016 Rohypnol, 3, 28 (XXIX) (flunitrazepam) has been successfully determined19 in of 29 white “cappuccino” style coffee by LC-DED by a novel dual reductive mode approach [20]. Studies were performedRecently, to optimise Rohypnol the chromatographic (XXIX) (flunitrazepam) conditions has been and were successfully reported determined to be 50% in acetonitrile, white 50% 50“cappuccino” mM pH 2.0style phosphate coffee by LC buffer-DED atby aa flownovel ratedual ofreductive 0.75 mL/min, mode approach employing [20]. Studies a Hypersil were C18, 5 mm,performed 250 mm to× optimise4.6 mm the column. chromatographic Cyclic voltammetric conditions and studies were reported were madeto be 50 to% acetonitrile, ascertain the 50% redox behaviour50 mM of pH Rohypnol 2.0 phosphate (XXIX) buffer at a glassyat a flow carbon rate of electrode 0.75 mL/min, over employing the pH range a Hypersil 2–12. HydrodynamicC18, 5 mm, 250 mm × 4.6 mm column. Cyclic voltammetric studies were made to ascertain the redox behaviour voltammetry was used to optimise the applied potential at the generator and detector cells; these of Rohypnol (XXIX) at a glassy carbon electrode over the pH range 2–12. Hydrodynamic voltammetry were identified to be −2.4 V and +0.8 V for the redox mode and −2.4 V and −0.1 V for the dual was used to optimise the applied potential at the generator and detector cells; these were identified reductiveto be mode −2.4 V respectively. and +0.8 V for A linear the redox range mode of 0.5–100 and −2.4 mg/mL, V and with−0.1 V a detection for the dual limit reductive of 20 ng/mL mode was obtainedrespectively. for thedual A linear reductive range of mode. 0.5–100 Further mg/mL, studies with a weredetection then limit performed of 20 ng/mL to identify was obtained the optimum for conditionsthe dual required reductive for mode. the LC-DED Further determinationstudies were then of performed Rohypnol to (XXIX) identify in beveragethe optimum samples. conditions In order to demonstraterequired for the the application LC-DED determination of the LC-DED of Rohypnol assay to (XXIX)forensic in “drink beverage spiking” samples. cases In order a sample to of whitedemonstrate cappuccino the style application coffee was of the fortified LC-DED at assay a level to forensic of 9.6 µ “drinkg/mL spiking” Rohypnol cases (XXIX). a sample Figure of white4 shows the LC-DEDcappuccino chromatograms style coffee was forfortified extracts at a level of (i) of coffee 9.6 µg/mL spiked Rohypnol with 9.6 (XXIX).µg/mL Figure and 4 shows (ii) for the unspiked LC- coffee.DED Clearly, chromatograms when using for extracts the dual of reductive(i) coffee spiked mode with the extracts9.6 µg/mL showed and (ii) well-defined for unspiked signalscoffee. for Clearly, when using the dual reductive mode the extracts showed well-defined signals for Rohypnol Rohypnol (XXIX). However, when using the redox mode the region from a retention time of 11 min (XXIX). However, when using the redox mode the region from a retention time of 11 min onwards is onwards is totally obscured by a large off-scale unresolved peak, which completely masks the area totally obscured by a large off-scale unresolved peak, which completely masks the area where whereRohypnol Rohypnol (XXIX) (XXIX) elutes. elutes. The Thedual dual reductive reductive mode modewas hence was hencereported reported to give toreliable give reliabledata at the data at the concentrationsconcentrations investigatedinvestigated relevant relevant to tocases cases of drink of drink spiking. spiking.

Figure 4. Representative chromatograms of “cappuccino” style white coffee samples obtained by Figure 4. Representative chromatograms of “cappuccino” style white coffee samples obtained by LC-DED in the redox mode for (i) fortified at 9.6 µg/mL (ii) LC-DED dual reductive mode, fortified LC-DED in the redox mode for (i) fortified at 9.6 µg/mL (ii) LC-DED dual reductive mode, fortified at at 9.6 µg/mL and (iii) unadulterated. R = Rohypnol (XXIX). 9.6 µg/mL and (iii) unadulterated. R = Rohypnol (XXIX). 3.5. Amphetamines 3.5. Amphetamines Amphetamines can be difficult to determine electrochemically requiring high positive Amphetaminesapplied potentials can for be their difficult determination. to determine To overcome electrochemically this problem, a requiring derivatization high using positive applied2,5-d potentialsihydroxybenzaldehyde for their determination. (2,5-DBA) has been To described overcome by this Alfredo problem, Santagati a derivatizationet al. [69]. It was using 2,5-dihydroxybenzaldehydeshown that 2,5-DBA could (2,5-DBA) rapidly has aminated been described the primary by Alfredo amines ofSantagati amphetamine et al. [69 (XXX),]. It was 4-hydroxynorephedrine (XXXI) and phenylethylamine (XXXII) (PHE), using borohydride exchange shown that 2,5-DBA could rapidly aminated the primary amines of amphetamine (XXX), resin as a chemoselective giving electroactive secondary amines. LC ED analysis was 4-hydroxynorephedrineperformed using reversed (XXXI) phase and isocratic phenylethylamine elution on a column (XXXII) 5 µm (PHE), Hypersil using ODS borohydride RP-18, 15 cm, exchangewith resina as mobile a chemoselective phase of methanol reducing-NaH agent2PO4 buffer giving (50 electroactive mM, pH 5.5) secondary (30:70 v/v) amines. containing LC triethylamine ED analysis was performed(0.5% usingv/v). The reversed electrochemical phase isocratic detection elution of on the a columnderivatised 5 µ m compounds Hypersil ODS was RP-18, investigated 15 cm, by with a mobilehydrodynamic phase of methanol-NaH voltammetry2 PO at 4 abuffer porous (50 graphite mM, pH electrode 5.5) (30:70 andv/ v) under containing the chromatographic triethylamine (0.5% v/v).conditions The electrochemical employed the detection optimum of potential the derivatised was reported compounds to be +0.6 was V. The investigated linearity of by response hydrodynamic was voltammetryexamined at for a each porous derivatised graphite compound electrode and and was under analysed the chromatographic using solutions in conditionsthe range 10employed to 40 the optimumnM/mL. The potential correlation was coefficients reported toof the be +0.6linear V. regression The linearity of the ofstandard response curves was were examined greater than for each derivatised compound and was analysed using solutions in the range 10 to 40 nM/mL. The correlation Separations 2016, 3, 28 20 of 29 coefficients of the linear regression of the standard curves were greater than 0.99. A detection limit Separations 2016, 3, 28 20 of 29 basedSeparations on a signal/noise 2016, 3, 28 ratio of 3:1 (S/N = 3) was less than 50 ng/mL for each compound20 of and 29 the limits of quantitation0.99. A detection were limit comprised based on a signal/noise in the range ratio 0.3–0.6of 3:1 (S/Nµg/mL. = 3) was less than 50 ng/mL for each 0.99. compoundA detection and limit the limitsbased of on quantitation a signal/noise were comprisedratio of 3:1 in (S/N the range = 3) was0.3–0.6 less µg/m thanL. 50 ng/mL for each compound and the limits of quantitation were comprised in the range 0.3–0.6 µg/mL.

(XXX) (XXXIII)

(XXX) (XXXIII)

(XXXII)

Kramer and Kovar [70] have determined N-ethyl-4-hydroxy-3-methoxy -amphetamine (XXXIII) Kramer(HMEA, and the Kovar main [metabolite70] have of determined the ecstasy analogueN(XXXII)-ethyl-4-hydroxy-3-methoxy-amphetamine MDE), THC and THC–COOH in plasma and (XXXIII) (HMEA, theurine main utilising metabolite automated of on the-line ecstasy SPE. Liquid analogue chromatographic MDE), THC separation and THC–COOHwas achieved using in plasma a and urine utilisingKramerLiChroC automated artand Superspher Kovar [ on-line70 60] haveRP-select SPE.determined B Liquidcolumn, N 5 chromatographic- µm,ethyl 250-4 -×hydroxy 4 mm with-3- methoxya separation LiChrospher-amphetamine was60 RP achieved-select (XXXIII) B, using a LiChroCart(HMEA,5 µm, Superspher the 4 ×main 4 mm metabolite guard 60 RP-select column. of the HMEA B ecstasy column, was analoguedetermined 5 µm, 250MDE), using× 4 aTHC mmmobile and with phase THC a LiChrospherconsisted–COOH of in 150 plasma 60mM RP-select and B, potassium dihydrogen phosphate buffer, pH 2.3–acetonitrile (94.5:5.5, v/v) with 0.16 mM Na EDTA, 5 µm,urine 4 × utilising4 mm automated guard column. on-line SPE. HMEA Liquid was chromatographic determined using separation a mobile was achieved phase consisted using a of LiChroCbufferart– acetonitrileSuperspher mixture 60 RP -select with aB flowcolumn, rate 5 ofµm, 0.600 250 mL/min.× 4 mm with Electrochemical a LiChrospher detection 60 RP was-select B, 150 mM potassiumundertaken dihydrogenusing a potential phosphate of +920 mV. buffer, THC and pH THC 2.3–acetonitrile–COOH were separated (94.5:5.5, isocraticallyv/v) with using 0.16 mM Na 5 µm, 4 × 4 mm guard column. HMEA was determined using a mobile phase consisted of 150 mM EDTA, buffer–acetonitrilea LiChroCart, Superspher mixture 60 RP with select aB flowcolumn, rate 5 µ ofm, 0.600250 mm mL/min. × 4 mm. The Electrochemical mobile phase consisted detection was potassium dihydrogen phosphate buffer, pH 2.3–acetonitrile (94.5:5.5, v/v) with 0.16 mM Na EDTA, undertakenof 5.6 using mM atetrabutylammonium potential of +920 hydrogen mV. THC sulphate, and THC–COOH pH 2.3–acetonitrile were–tetrahydrofuran separated isocratically (44:46:10) using a bufferwith–acetonitrile 0.160 mM mixture Na EDTA. with The a flow flow-rate rate in of this 0.600 case mL/min. was reported Electrochemical as 0.850 mL/min detection with was LiChroCart,undertakenelectrochemical Superspher using a detectionpotential 60 RP selectagain of +920 at B +1.2 column,mV. V. THC The 5limits andµm, THCof 250 quantitation– mmCOOH× 4 were were mm. separatedreported The mobile to isocraticallybe between phase consisted5 using of 5.6 mMa LiChroCart, tetrabutylammoniumng/mL (THC, Superspher THC–COOH 60 hydrogen RP in plasma) select B sulphate, and column, 20 ng/mL 5 pH µ (HMEAm 2.3–acetonitrile–tetrahydrofuran, 250 mmin plasma). × 4 mm. The mobile phase (44:46:10) consisted with 0.160of mM 5.6 NamM EDTA.tetrabutylammonium The flow-rate hydrogen in this case sulphate, was reported pH 2.3–acetonitrile as 0.850 mL/min–tetrahydrofuran with electrochemical (44:46:10) detectionwith again 0.160 mMat +1.2 Na V. EDTA. Thelimits The flow of quantitation-rate in this were case reportedwas reported to be as between 0.850 mL/min 5 ng/mL with (THC, THC–COOHelectrochemical in plasma) detection and again 20 ng/mL at +1.2 (HMEA V. The limits in plasma). of quantitation were reported to be between 5 ng/mL (THC, THC–COOH in plasma) and 20 ng/mL (HMEA in plasma).

3.6. Legal Highs: Mephedrone and 4-Methylethcathinone Legal highs can be defined as drugs that contain one or more chemical substances which produce similar effects to illegal drugs. These are quite often made by changing functional groups on already developed drugs to give a new substance for which there is no specific legalisation. Consequently, these new compounds are not controlled under the Misuse of Drugs Act 1971 and often there is insufficient investigations undertaken on their potency, adverse effects, or interactions with other 3.6. Legal3.6. Legal Highs: Highs: Mephedrone Mephedrone and and 4-Methylethcathinone 4-Methylethcathinone substances. This is a rapidly changing area with new drugs being created and sold over the Internet. LegalLegal highsZuway highs can etcan be al. be defined [71 defined] have as recently as drugs drugs investigated that that containcontain the one onepossibility or or more more of chemical determining chemical substances substancescathinone which-derived which produce produce similarlegal effects highs to byillegal liquid drugs. chromatography These are quite with often amperometric made by changing detection forfunctional the determination groups on ofalready similar effects to illegal drugs. These are quite often made by changing functional groups on already developedmephedrone drugs (XXXIV)to give a and new 4 -substancemethylethcathinone for which (XXXV). there isReverse no specific phase legalisation. chromatography Consequently, was developed drugs to give a new substance for which there is no specific legalisation. Consequently, theseundertaken new compounds using an aACEre not 3 C controlled18, 150 mm × under 4.6 mm, the 3 Misuseμm column of Drugswith mobile Act 1971phase and of methanol: often there is 10 mM ammonium acetate–100 mM potassium chloride buffer, 30%:70% v/v using a flow rate of either theseinsufficient new compounds investigations are not undertaken controlled on undertheir potency, the Misuse adverse of effects, Drugs or Act interactions 1971 and with often other there is insufficientsubstances. investigations This is a rapidly undertaken changing onarea their with potency,new drugs adverse being created effects, and or sold interactions over the Internet. with other substances.Zuway This et is al. a rapidly [71] have changing recently areainvestigated with new the drugs possibility being of created determining and soldcathinone over- thederived Internet. Zuwaylegal highs et al. by [71 liquid] have chromatography recently investigated with the amperometric possibility ofdetection determining for the cathinone-derived determination of legal highsmephedrone by liquid chromatography (XXXIV) and 4-methylethcathinone with amperometric (XXXV). detection Reverse for the phase determination chromatography of mephedrone was (XXXIV)undertaken and 4-methylethcathinone using an ACE 3 C18, 150 (XXXV). mm × 4.6 Reverse mm, 3 phase μm with mobile phase was undertaken of methanol: using 10 mM ammonium acetate–100 mM potassium chloride buffer, 30%:70% v/v using a flow rate of either an ACE 3 C18, 150 mm × 4.6 mm, 3 µm column with mobile phase of methanol: 10 mM ammonium

Separations 2016, 3, 28 21 of 29 acetate–100 mM potassium chloride buffer, 30%:70% v/v using a flow rate of either 0.8 mL/min or 1.0 mL/min. Four different thin-layer flow cells investigated each employing a screen-printed sensor for the amperometric detection of the two drugs at an applied potential of +1.4 V. The effect of flow rate was investigated and improvements in sensitive were found at 0.8 mL/min compared to 1.0 mL/min. Detection limits of 14.66 and 9.35 µg/mL for mephedrone (XXXIV) and 4-methylethcathinone (XXXV) were reported. Analysis of the synthetic cathinones in a selection of purchased NRG-2 legal high samples was undertaken. In samples containing caffeine UV and amperometric determination were found to be comparable in terms of their ability to quantify the levels of caffeine present. Samples containing only mephedrone (XXXIV) and 4-methylethcathinone (XXXV) showed a significant over estimation of the quantities of the synthetic cathinones present in comparison to the HPLC-UV detection. The authors concluded that this may be due to adsorption of the drugs on the electrode surface and a new screen-printed sensor was employed for each sample analysis to overcome this. The developed liquid chromatographic electrochemical method was found to be less sensitive than the liquid chromatography with UV detection.

3.7. Tryptamines, Phenethylamines and Piperazines To restrict the sale of legal highs in April 2007 Japan introduced the Pharmaceutical Affairs Law where substances such as tryptamines, phenethylamines and piperazines became under control as ‘designated substances’ (Shitei-Yakubutsu). The relative large number of compounds required a technique which was capable of separating, identifying and quantifying such compounds in complex samples. To achieve this Min et al. [72] developed a liquid chromatographic multichannel electrochemical detection (MECD) method for the simultaneous determination of 31 different tryptamines, phenethylamines and piperazines. The compounds were separated by reverse phase chromatography using a gradient elution. A coulometric electrode array detector (Model 5600A CoulArray, ESA Inc., (Thermo Scientific, Waltham, MA, USA) equipped with 16 channel cell electrodes (model 6210, porous graphite working electrode) was used for the detection. The optimum applied potential for each substance was determined based by hydrodynamic voltammetry. The mobile phases A and B consisted of 31.4 mM potassium phosphate buffer–methanol–acetonitrile (95:4:1; pH 6.7) and 60 mM potassium phosphate buffer–methanol–acetonitrile (50:40:10; pH 6.7), respectively. Separation was performed using a reversed-phase ODS column (TSK-gel ODS-100V, 250 × 4.6 mm, 3 µm) with gradient elution of 25% B (0–20 min), 10% B (20–60 min) and 10%–70% B (60–240 min) at the flow rate of 1.0 mL/min. The applied potentials of the 16 channel electrodes were set at 0, 90, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260 and 1350 mV. Detection limits (S/N = 3) ranged from 17.1 pg N-[2-(5-methoxy-1H-indol-3-yl)ethyl]-N-methylpropan-2-amine (XXXVI) (5-MeO-MIPT) to 117 ng 2,3-dihydro-1H-inden-2-amine (XXXVII) (indan-2-amine). The developed method was evaluated by the analysis of real samples. Solid samples (1 mg) were dissolved in 1.0 mL of a 50% methanol with the aid of sonication and then centrifuged. The separated supernatant was filtered and the solutions diluted 100 times with the initial mobile-phase starting solution and injected into the HPLC-MECD. The target analytes were identified by their retention times and the hydrodynamic voltammograms of authentic standards. Table3 presents a summary of the applications discussed in this section.

1

Separations 2016, 3, 28 22 of 29

Table 3. Liquid chromatography electrochemical determination of synthetic drugs.

Analyte LC ED Technique Reference Electrode Linear Range Detection Limit Comments Ref. Dual amperometric Rohypnol reductive-reductive mode; Stainless (generator cell); 0.5–100 mg/mL 20 ng/mL Bovine and human serum [20] (flunitrazepam) electrode 1; −2.4 V and Ag/AgCl (detector cell) electrode 2; −0.2 V Amphetamine and Porous graphite electrode; 50 ng/mL for each Derivatisation with PdH 10–40 nM/mL [69] 4-hydroxynorephedrine +0.6 V. 2 compound 2,5-dihydroxybenzaldehyde Mephedrone; Mephedrone and Screen-printed carbon 14.66 µg/mL; Samples of the ‘legal high’ Screen-printed Ag/AgCl [71] 4-methylethcathinone electrode; +1.4 V 4-methylethcathinone NRG-2 analysed. 9.35 µg/mL. Tryptamines, Ranged from 17.1 pg Multichannel 31 different drugs Phenethylamines and PdH to 117 ng [72] electrochemical detection 2 determined Piperazines indan-2-amine Separations 2016, 3, 28 23 of 29

Separations 2016, 3, 28 23 of 29

(XXXVI)

(XXXVII)

4. Comparisons Comparisons with with Other Other Liquid Liquid Chromatographic Chromatographic Detection Detection Systems Systems Principally, two other classes of detector have been utilized with liquid chromatography: those based on the absorbance of of light light in in some some way, way, such as as UV UV-visible-visible and fluorescence fluorescence spectrometry and those using using mass mass spectrometry. spectrometry. Liquid Liquid chromatography chromatography mass mass spectrometry spectrometry is now is widely now widely used across used acrossindustry, , in research in research and the and forensic the forensic sciences. sciences. It can It be can extremely be extremely selective selective and and sensitive sensitive and and can canbe successfully be successfully used used for for a wide a wide range range of of analytes. analytes. Confirmation Confirmation of of peak peak identityidentity obtainedobtained through the mass spectra obtained is is also also highly highly useful. useful. However, However, it is relatively expensive and needs highly trained staff. As a technique, LC/MS can can s sufferuffer from from issues issues with with selectivity selectivity resulting resulting from from “isobaric” “isobaric” interferences, unpredictable yield attenuations from “ion suppression effects” [73]. Some of these issues can be be overcome overcome by by the the use use of of deuterated deuterated standards; standards; however, however, these can be expensive. expensive. Table Table 44 gives aa comparisoncomparison between between the the detection detection limits limits reported reported for bothfor both LC/MS LC/MS and LCand ED LC for ED several for several drugs drugsof abuse. of abuse. For anumber For a number of compounds of compounds detection detection limits are limits comparable are comparable and in someand in instance some instance notably notablybetter by better LC ED; by however,LC ED; however, as a general as a approachgeneral approach LC/MS canLC/ beMS seen can tobe beseen better to be across better the across range the of rangeanalytes of investigated.analytes investigated. Liquid chromatography Liquid chromatography with UV detection with UV (LC-UV) detection is both(LC-UV) simple is both and reliable,simple andbut lacksreliable, the lowbut lacks detection the low limits detection that can limits be gained that bycan both be gained LC ED by and both LC/MS LC ED (Table and4 ).LC/MS Advantages (Table 4can). Advantages be gained using can be variants gained suchusing as variants diode array such detectionas diode array (DAD) detection where spectra (DAD) can where be obtained spectra can for beeach obtained eluting peakfor each and eluting can hence peak aid and in peak can hence identification aid in peak and questionsidentification of peak and purity. questions LC ED of offerspeak purity.comparable, LC ED or offers in some comparable, cases better or detection in some limitscases andbetter is considerablydetection limits less and expensive is considerably than LC/MS. less expensiveHowever, itthan can LC/MS. be affected However, by fouling it can of thebe affected electrodes by leading fouling to of loss the ofelectrodes sensitivity. leading The presence to loss of sensitivity. and The presence in of the oxygen mobile and phase metal can beions an in issue the mobile as well, phase but can can be be overcome an issue by as degassingwell, but canand bethe overcome addition of by a chelating agent, and approachesthe addition that of are a chelating both commonly agent, approachesemployed. that are both commonly employed.

Table 4. Comparisons with Liquid Chromatography Mass Spectrometry.

LC ED Detection LC/MS Detection LC/UV Detection Analyte Ref. Ref. Ref. Limit, ng/mL Limit, ng/mL Limit, ng/mL THC 0.5 [38] 1.0 [74] 746 [75]

methadone 0.9 [54] 0.1 [76] buprenorphine 0.08 [54] 5.0 [76] 20 [77] norbuprenorphine 0.08 [54] 1.0 [76] morphine 0.5 [47] 0.5 [76] 10 [47]

Separations 2016, 3, 28 24 of 29

Table 4. Comparisons with Liquid Chromatography Mass Spectrometry.

LC ED Detection LC/MS Detection LC/UV Detection Analyte Ref. Ref. Ref. Limit, ng/mL Limit, ng/mL Limit, ng/mL THC 0.5 [38] 1.0 [74] 746 [75] methadone 0.9 [54] 0.1 [76] buprenorphine 0.08 [54] 5.0 [76] 20 [77] norbuprenorphine 0.08 [54] 1.0 [76] morphine 0.5 [47] 0.5 [76] 10 [47] codeine 24 [55] 1.0 [76] 6.0 [78] amphetamine <50 [69] 0.25 [76] 100 [79] mephedrone 14,660 [71] 5.0 [80] 803 [81] 4-Methylethcathinone 9350 [71] 5.0 [80] 2410 [71] psilocybin 0.8 [67] 2.0 [82] 50 [83] Rohypnol 20 [84] 0.2 [82] 30 [85] nitrazepam 100 [20] 1.25 [86]

5. Conclusions The combination of liquid chromatography with electrochemical has been shown to a powerful technique for the determination of drugs of abuse capable of determining trace concentration in complex sample matrixes. The detection of morphine has seen the most interest presumably due to the low applied potential required for its oxidation and it demand for its determination in both and forensic applications. Applications for new classes of illegal drugs are being developed. However, the methods developed for the determination of the more established drugs of abuse are generally depleted, preassembly, due to the introduction of competing techniques such as LC/MS. The majority of methods have been based on reverse phase chromatography. However, it is believed that more work will be reported on the application of techniques such as hydrophilic interaction chromatography (HILIC) [87]. Similarly, the combination of electrochemistry with mass spectrometry and LC/MS [88] will be become an increasingly important area for drug studies.

Acknowledgments: I would like to thank my fellow researchers whose work has been described in this review. Conflicts of Interest: The author declares no conflicts of interest.

Abbreviations The following abbreviations are used in this manuscript: 2,5-DBA 2,5-dihydroxybenzaldehyde 4HIAA 4-hydroxyindole-3-acetic acid 5-MeO-MIPT N-[2-(5-methoxy-1H-indol-3-yl)ethyl]-N-methylpropan-2-amine Ag/AgCl / BAS Bioanalytical Systems, Inc. EtG Ethyl glucuronide GCE Glassy carbon electrode GC/MS Gas chromatography mass spectrometry HILIC Hydrophilic interaction chromatography HMEA N-ethyl-4-hydroxy-3-methoxy-amphetamine HPLC EA High performance liquid chromatography electrochemical array detection HPLC-UV High performance liquid chromatography-ultra violet detection IDU Intravenous drug user LC-DED Liquid chromatography dual electrode detection LC ED Liquid chromatography electrochemical detection LC/MS Liquid chromatography mass spectrometry LC-UV Liquid chromatography ultraviolet detection LSD Lysergic acid diethylamide MDE Methylenedioxyethylamphetamine MECD Multichannel electrochemical detection Na2EDTA Disodium ethylenediaminetetraacetic acid Separations 2016, 3, 28 25 of 29

NRG-2 4-Methylethcathinone legal high marketed alone or in mixtures with other substituted cathinones PBS Phosphate buffered saline PdH2 Palladium-hydrogen reference electrode PED Pulsed electrochemical detection PIA Pulsed integrated amperometry PHE Phenylethylamine S/N Signal-to-noise ratio SPE Solid-phase extraction THC Tetrahydrocannabinol THC–COOH 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid TLC Thin-layer cell

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