A number of different chromatographic approaches have been employed for the determination of cannabis and its constituent cannabinoids and have been reviewed [32
]. HPLC-UV procedures are widely used for the analyses of the cannabinoids utilising wavelengths between 215 and 280 nm. However, the sensitivity of the electrochemical detector has been shown to be over 400 times greater than that obtained by UV at 220 nm [33
]. Around 70 different cannabinoid compounds are reported present in herbal cannabis [34
], however, Δ9
-Tetrahydrocannabinol (I) (THC) is the major psychoactive component and methods for its determination are consequently more commonly reported. Nyoni et al. [35
] have developed a LC ED based method to assist in investigating the biochemical mechanism(s) of THC (I) in brain tissue. The method employed solvent extraction with methanol-hexane-ethyl acetate, followed by analysis using LC ED. Overall recoveries were reported to be greater than 80%.
Bourquin and Brenneisen [36
] utilised reverse phase LC ED for the determination of the major metabolite of THC, 11-nor-Δ9
-tetrahydrocannabinol-9-carboxylic acid (II) (THC–COOH) in urine. Separation was achieved using a mobile phase of methanol-5% aqueous acetic acid (76:24) at a flow-rate of 1.5 mL/min. The detector was operated in the amperometric mode, at an applied potential of +1.2 V (vs. Ag/AgCl). A 10 mL aliquot of human urine was fortified with internal standard (cannabinol) and hydrolysed with KOH by heating to convert the excreted glucuronide conjugates to free, unconjugated THC–COOH (II). The sample pH was then adjusted to pH 5–6 and the internal standard and THC–COOH (II) isolated by solid phase extraction (SPE) [37
]. Both THC–COOH (II) and the internal standard were 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 calibration curve was obtained by using blank urine spiked with 25–300 ng/mL THC–COOH (II) and 90 ng/mL internal standard.
Further investigations [38
] into the simultaneous determination of THC and the further metabolites THC–COOH and 11-hydroxy-Δ9
-tetrahydrocannabinol (III) (11-OH THC) in urine and plasma by LC ED have been reported. Biological fluids were first hydrolysed by heating in base and then acidified and extracted with a previously described automatic extractor [39
] and determined by LC ED with amperometric determination at +1.1 V. Liquid chromatography was carried out with a reversed-phase silica C8
column and a mobile phase of acetonitrile/methanol/0.02 N H2
(35:15:50) at a flow rate of 1.8 mL/min. A detection limit of under 0.5 ng/mL (S/N > 3) with a linear range of 10–500 ng/mL was reported. Using this approach it was found possible to detect THC–COOH (II) in rabbit urine up to 216 h after administration. Investigations were also made on samples of human urine obtained from 23 men arrested on the suspicion of smoking marijuana and results showed good agreement with those obtained by GC/MS [40
THC has also been determined in human urine using a novel brominated 9-carboxy-11–nor-Δ9
-tetrahydrocannabinol as an internal standard [41
]. Amperometric determination was undertaken at +0.85 V (vs. Ag/AgCl) using gradient elution. Problems generally exist with the application of gradient elution when used in conjugation with LC ED. Electrochemical detector background current is a function of organic solvent composition and ionic strength. Consequently, the application of solvent gradient will result in a continually changing background current. However, the authors showed that this can be minimized by making the ionic strengths of the two components of the mobile phase identical. Using this approach the authors reported only a small shift in the baseline of around 1 to 2 nA. After addition of internal standard samples of human urine were adjusted to be between pH 4.5 and pH 6.5 and isolated by SPE. No interferences were reported for 22 drugs and metabolites. A pooled relative standard deviation of 4.1% (n
= 27) was obtained for the quality control samples and the method showed good agreement with results obtained by gas chromatography/mass spectrometry.
Nakahara and Tanaka [42
] have develop a chemotaxonomical based LC ED method for the discrimination of confiscated cannabis samples. Eleven different samples were investigated and on the basis of their liquid chromatographic cannabinoid profiles. They demonstrated the possibility of distinguishing cannabis products grown in three districts of Japan, Colombia, The Philippines and Nepal. The stability of cannabinoids in cannabis products was also examined at room temperature, 4 °C and −20 °C. After 16 months, it was found that 90% of original THC (I) remained at −20 °C, 50% and 28% of original THC (I) had decomposed at room temperature and 4 °C, respectively. Almost all of the decomposed THC (I) had changed to cannabinol by air oxidation.
Nakahara and Sekine [42
] have used LC ED for the determination of free cannabinoids and cannabinoic acids obtained from marijuana cigarettes and in tar and ash obtained by using an automatic smoking machine. Separation was achieved using reverse phase chromatography with mobile phase of 0.02 N H2
, methanol acetonitrile (6:7:16) at a flow rate of 1.1 mL/min. Amperometric detection was undertaken using an applied potential of +1.2 V (vs. Ag/AgCl) and a liner range of 5 to 500 ng/injection was reported with detection limits of 0.5 to 0.9 ng/injection 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. [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 KH2
CN (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%. Table 1
gives a summary of the LC ED approaches reported for the determination of cannabinoid compounds.
Previously, Tagliaro et al. [9
] have reviewed the LC ED applications for the determination of morphine. Schwartz and David [46
] have explored the liquid chromatography electrochemical determination of morphine (VIII), heroin (IX), codeine (X), thebaine (XI), narcotine (XII), papaverine (XIII) and cocaine (XIV). Compounds were determined by reverse phase chromatography using an acetonitrile phosphate or acetate buffer based mobile phase with amperometric detection undertaken at +1.2 V. The mechanism for electrochemical oxidation of amines is known to involve the lone pair of electrons on the nitrogen atom [47
] the pH consequently should be high enough to maintain these compounds in their basic form. In aqueous solutions this would require a pH 10 or above. However, under the chromatographic conditions employed the authors were able to gain good sensitivity using lower pH values in the 6–8 range. The authors concluded that in the presence of the mobile phase organic modifier the tertiary nitrogen atom remains in its basic un-protonated form at pH values lower than that when strictly aqueous media are used. This enhancement of basicity was concluded to result from the organic solvent present in the mobile phase. Limits of detection were determined to be 0.3 ng for morphine, 1 ng for heroin, and 2 ng for cocaine.
Sawyer et al. [52
] have developed a method for the determination of, morphine (VIII), heroin (IX) and hydromorphone (XV) from post-mortem tissues. Post-mortem samples of whole blood, urine, or vitreous humour were assayed without pre-treatment. It was reported necessary to preserved samples with buffered sodium fluoride/sodium azide to prevent changes in the levels of heroin (IX) and morphine (VIII). One-hundred µL sample aliquots were taken and the internal standard, nalorphine, and ammonium chloride/ammonium hydroxide buffer added. These were then extracted with 5 mL of dichloromethane:isopropanol (96:4 v
). Following centrifugation the organic phase was extracted with pH 3 phosphate-citrate buffer and the solvent discarded. The resulting aqueous layer was then extracted with pH 8.50 buffer and dichloromethane:isopropanol (96:4 v
). The organic was then taken and evaporated to dryness under nitrogen and reconstituted in mobile phase and examined by LC ED. A single-step extraction procedure was also developed using a 99.5:0.5 dichloromethane isopropanol solution. However, due to interferences from endogenous metabolites in urine or other commonly encountered drugs was not recommended.
Hydrodynamic voltammetry was employed to explore the electrochemical behaviour of the three target analytes under reverse phase 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 of +0.5 V was reported to be the optimum applied potential for use with a Bioanalytical Systems, Inc. (BAS, West Lafayette, IN, USA) LC-3 single cell amperometric detector. Linear responses were recorded from 10 to 500 ng/mL morphine (VIII), 62 to 1000 ng/mL hydromorphone (XV), and 250 to 2000 ng/mL 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 drugs were investigated as possible interferences. Out of theses only acetaminophen, aminopyrine, cyanide, disopyramide, ketamine and nalorphine were found to give chromatographic peaks, but were reported not to interfere. Material from six human post-mortem cases of suspected heroin related death were examined and the concentrations of heroin (IX) and morphine (VIII) determined in both blood and urine showed good agreement with those obtained by radioimmunoassay.
Zaromb et al. [53
] have investigated the determination of airborne cocaine (XIV) and heroin (IX) using high-throughput liquid adsorption pre-concentration. Air was sampled at a rate over the range 550–700 L/min with a preferred ranged of 620–680 L/min. Detection limits of ca. 1:1013
) of the drugs were achieved. LC ED was undertaken using a mobile phase of potassium phosphate buffer (pH 7–7.4, 0.02 M)-acetonitrile (40:60, v
) at a flow rate of 1.0 mL/min, at C18
stationary phase. Amperometric detection was undertaken at a glassy carbon electrode at an applied potential of +1.0 V (vs. Ag/AgCl).
Buprenorphine (XVII), naloxone (XVIII) (Suboxone®
) and methadone (XIX) are commonly used for the treatment of opioid addiction. Somaini et al. [54
] have reported a LC ED coulometric based method for their determination in human blood plasma utilising levosulpiride as an internal standard. Chromatographic separation was achieved using a mobile phase of 40:60, v
acetonitrile-2.5 mM pH 6.4 phosphate buffer at a flow rate of 0.6 mL/min with a cyano 250 mm × 3.0 mm, 5 µm column. Dual electrode detection was undertaken with the conditioning cell set at +0.05 V; the upstream screening cell set at −0.20 V and the detector electrode at +0.60 V. A rapid clean-up procedure of the biological samples using a microextraction by packed sorbent technique was also reported, employing C8
sorbent inserted into a syringe needle. Calibration curves were reported to be linear over a range of 0.25–20.0 ng/mL for buprenorphine (XVII) and norbuprenorphine (XX), 3.0–1000.0 ng/mL for methadone (XIX) and 0.13–10.0 ng/mL for naloxone with detection limits of 0.08 ng/mL for both buprenorphine (XVII) and norbuprenorphine (XX), 0.9 ng/mL for methadone (XIX) and 0.04 ng/mL for naloxone (XVIII). The method was successfully applied to plasma samples obtained from former heroin addicts treated with opioid replacement therapy.
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 filtering with these cottons is prevalent and part of the drug paraphernalia used by intravenous drug users (IDUs) in their injection ritual, they represent an ideal source for obtaining samples for analysis. Huettl et al. have reported a high performance liquid chromatography electrochemical array detection (HPLC-EA) [55
] method for the determination of heroin (IX), morphine (VIII), codeine (X) and cocaine (XIV) contained in these drug cottons. Two different extraction methods were investigated, using either water or ethyl acetate to extract the drugs from the cotton filters. Following a 10 min incubated at room temperature with the selected solvent, the samples were centrifuged and the supernatant taken and either blown down to dryness in the case of ethyl acetate or in the case of water, diluted in mobile phase and filtered before examination by HPLC-EA. Due to the heterogeneous nature of the drug cotton samples standard addition was employed to verify the retention time of the target analytes heroin (IX), morphine (VIII), codeine (X) and cocaine (XIV). Separation and analysis of the extracts were carried out using a CoulArray gradient HPLC coupled to a twelve coulometric electrochemical array detector arranged in series after the analytical column. The potentials of each of the electrodes in the array was set at increasing potentials of (1) 300 mV; (2) 350 mV; (3) 400 mV; (4) 450 mV; (5) 500 mV; (6) 550 mV; (7) 600 mV; (8) 650 mV; (9) 700 mV; (10) 750 mV; (11) 850 mV and (12) 950 mV. Sample separation was accomplished using an end-capped cyano (100 mm × 4.6 mm, 5 µm, CPS-2 Hypersil) column (Thermo Scientific, Waltham, MA, USA) with gradient elution. Initial conditions of 95% (A) 30 mM dibasic sodium phosphate, pH 2.95, 5% (B) 30 mM dibasic sodium phosphate, 30% ethanol, pH 2.95 at a flow rate of 1 mL/min for 5 min. The concentration of component (B) was then ramped to 80% over a period of 30 min. Detection limits of the HPLC-EA system were reported as: 4 pg/µL for morphine (VIII), 24 pg/µL for codeine (X), 444 pg/µL, heroin (IX) and 576 pg/µL for cocaine (XIV). The authors reported that it would be possible by using a slightly modified method to determine lysergic acid diethylamide (XXI) (LSD) and the THC metabolites: 11-hydroxy-Δ9
-THC) and 11-nor-Δ9
-tetrahydrocannabinol-9-carboxylic acid (11-nor-Δ9
-THC-9-COOH). Investigations were also made into the possibility to associate a particular chromatographic profile with different cuts of injectable drugs. Drug cottons were obtained from different areas 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.
Using liquid chromatography dual electrode detection (LC-DED) in conjunction with UV detection Ary and Róna [56
] have reported on the determination of morphine (VIII) (7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol) and its glucuronides; morphine-3-glucuronide, morphine-6-glucuronide. Morphine (VIII) and its glucuronides were extracted from human plasma using Bond-Elut C18
(1 mL) SPE cartridges. Average extraction efficiencies of morphine (VIII), morphine-3-glucronitide and morphine-6-glucronitide were 95.4%, 96.1% and 96.8%, respectively. Separations were achieved using a Supelcosi LC-8DB reverse phase column (Sigma-Aldrich, St. Louis, MO, USA) with a mobile phase of 0.1 M KH2
(pH 2.5)-acetonitrile-methanol (94:5:1 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 antibody solid-phase extraction columns were fabricated from aldehyde-activated silica modified with polyclonal antibodies 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 K3
and 1.0 mM CoCl2
in 0.5 M KCl. The electrochemical properties of the cobalt hexacyanoferrate film modified GCE were investigated by cyclic voltammetry using 0.1 M phosphate buffered saline (pH 4.5) as the supporting electrolyte. 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+
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
) methanol-0.1 M phosphate-buffered saline (PBS, pH 4.5) which contained 1.0 × 10−4
EDTA 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
> 0.990) with a corresponding detection limit of 5.0 × 10−7
M (S/N of 3). The morphine (VIII) concentration in the brain dialysis 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
]. Most recently Aragon et al. [61
] have investigated the pharmacokinetics of 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 elution program consisted of 100% of this mobile phase mixture for 8 min, 85% mobile phase with 15% acetonitrile for 8–11 min and 100% mobile phase for 11–14 min. Separation was achieved with a 4.6 × 150 mm and 5 µm particle size column maintained at 40 °C. Dual electrode coulometric detection was undertaken using a guard cell +750 mV; electrode 1 +300 mV; electrode 2 +450 mV, with electrode 2 used for quantification. The retention times for morphine and morpine-6-glucronide were 7.5 and 5.5 min with limits of quantification of 7.8 ng/mL and 4 ng/mL respectively. Table 2
summaries reported LC ED application for the determination of alkaloids.
Fisher et al. [62
] have compared the serum concentrations of morphine after administration of a buccal tablet (25 mg) with those obtained after intramuscular injection (10 mg). Buccal morphine was administered to eleven healthy volunteers and intramuscular morphine was given to five preoperative surgical patients. Serum morphine concentrations were assayed by LC DED in samples taken up to 8 h after drug administration. Electrochemical detection was carried out using a 5100A Coulochem detector fitted with a 5100 detector cell (ESA). The potential of electrode 1 was maintained at +0.25 V and electrode 2 at +0.40 V. The lower limit of detection was reported to be 0.8 ng/mL morphine base. Morphine (VIII) analysis was carried out using the extraction and chromatographic procedure described by Todd et al. [63
]. Mean maximum morphine concentrations were eight times lower after buccal administration than after intramuscular injection and occurred at a mean of 4 h later. Individual morphine concentration-time profiles showed marked inter individual variability after administration of the buccal tablet, consistent with considerable variation in tablet persistence time on the buccal mucosa.
Jordan and Hart [64
] have investigated the determination of morphine (VIII) by liquid chromatography with amperometric determination at a GCE. Using cyclic voltammetry they showed that pH 11.0 was optimum for the electrochemical determination of morphine (VIII). Further investigations showed that a mobile phase of 50 mM pH 11.0 phosphate buffer of containing 20% v
of acetonitrile was found optimum. Hydrodynamic voltammetric investigations over the 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 +1.0 V. Using an applied potential of +0.45 V with hydromorphone (XXII) as an internal standard, a linear response was observed from 1.2 × 10−12
to 4 × 10−10
M of morphine injected. At the applied 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.
The hallucinogen psilocybin (XXIII) is one of the main psychoactive compounds found in Psilocybe
mushrooms. Psilocin (XXIV) is readily transformed to the phenolic compound psilocybin in the gut as a result of dephosphorylation by alkaline phosphatase and is thus the substance responsible for the reported psychedelic effects. Both psilocybin (XXIII) and psilocin (XXIV) are listed as Class A (United Kingdom) or Schedule I (US) drugs under the United Nations 1971 Convention on Psychotropic Substances. Interestingly, more recently, psilocin (XXIV) has been shown to have some promise as a therapeutic agent in the treatment of conditions cluster headaches [3
]. Lindenblatt et al. [66
] have used LC ED to determine levels of psilocin (XXIV) in plasma samples obtained from both pooled human blood bank plasma and plasma obtained from seven volunteers (self-experimenting physicians) taking oral doses of 0.2 mg psilocybin per kg body weight (maximum 15 mg per person) in a placebo-controlled drug trial. Both liquid-liquid and automated SPE procedures were explored for the isolation of psilocin (XXIV). The determination of psilocin (XXIV) obtained from liquid-liquid extracted samples was undertaken using reverse phase chromatography with a 250 × 4.0 mm C18
column and a mobile phase of 0.1 M sodium acetate, 0.1 M, citric acid, 0.03 mM Na2
EDTA pH 4.1–acetonitrile (83:17 v
, 0.7 mL/min) with electrochemical detection at +0.650 V using 5-hydroxyindole (XXV) as an internal standard. SPE extracts were determined using again, reverse phase chromatography but, with a mobile phase of 150 mM pH 2.3 potassium dihydrogen phosphate buffer–acetonitrile (94.5:5.5 v
, 0.6 mL/min) with 160 mM Na2
EDTA in the buffer–acetonitrile mixture. The potential of the electrochemical detector was set at +0.675 V with quantification carry out using bufotenine (XXVI) as an internal standard. The limit of quantitation for both methods was 10 ng/mL psilocin (XXIV). However, on-line SPE showed better recoveries and selectivity and reportedly required less manual effort and smaller plasma volumes of 400 µL, compared to 2 mL for liquid-liquid extraction.
A liquid chromatographic procedure based on column-switching with electrochemical detection has been developed for the determination of psilocin (XXIV) and the metabolite 4-hydroxyindole-3-acetic acid (XXVII) (4HIAA) in human plasma [67
]. Plasma was extracted from blood samples by centrifugation. Ascorbic acid was then added to protect the phenol compounds against degradation and the samples freeze-dried. The resulting residues were then re-constituted in water and the psilocin and 4HIAA (XXVII) extracted by microdialysis (mean recovery: psilocin 15.1% ± 0.85%; 4HIAA 11.0% ± 1.10%).
In order to avoid excess interfering plasma compounds such as ascorbic acid reaching the detector cell a column-switching step was employed. This was achieved by connecting the outlet of the injection valve was connected to a 5 cm Spherisorb RP-8 HPLC column allowing for pre-separation of the injected sample dialysate. The outlet of this pre-column was connected to the inlet of a second six-port Rheodyne valve; the flow of the eluate could be directed either to the waste or to the 15 cm Spherisorb RP-8 HPLC analytical column. The mobile phase consisted of 47% (v/v) water containing 0.3 M ammonium acetate buffered to pH 8.3 by addition of ammonia solution 25% and 53% (v/v) of methanol with a flow rate of 450 µL/min. Limits of quantification of 0.8 ng/mL and 5.0 ng/mL for psilocin (XXIV) and 4HIAA (XXVII) were reported respectively. The authors noted that optimised liquid chromatography UV detection of psilocin (XXIV) resulted in a limit of quantitation of approximately 10 ng/mL; allowing for measurement of only peak plasma concentrations. Similarly, GC/MS was reported to show insufficient sensitivity with the additional disadvantage of requiring a derivatising step.