Electrochemiluminescent sensors as a screening strategy for psychoactive substances within biological matrices.

With the rapid growth and appearance of novel psychoactive substances (NPS) onto the global drug market, the need for alternative screening methodologies for implementation within clinical environments is substantial. The immunoassay methods currently in use are inadequate for this new drug trend with the potential for misdiagnosis and subsequent administration of incorrect patient treatment increased. This contribution illustrates a strong proof-of-concept for the use of electrochemiluminescence (ECL) as a screening methodology for NPS within biological fluids, using the hallucinogen scopolamine as a model compound. A low cost, easy-to-use and portable sensor has been developed and successfully employed for the detection of scopolamine at clinically relevant concentrations within a variety of biological matrices, including human pooled serum, urine, artificial saliva and sweat, without any prior sample preparation required. Moreover, assessment of the sensor's potential as a point-of-care wearable device was performed with sample collection from the surface of skin, demonstrating its capability for the qualitative identification of scopolamine despite collection of only minimal volumes off the skins surface. The developed sensor described herein exhibits a strong proof-of-concept for the employment of such ECL sensors as point-of-care devices, where the sensors ease of use and removal of time-consuming and complex sample preparation methods will ultimately increase its usability by physicians, widening the avenues where ECL sensors could be employed.

Indirect competitive enzyme-linked immunosorbent assay based on a broad-spectrum monoclonal antibody for tropane alkaloids detection in pig urine, pork and cereal flours.

In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a broad-spectrum monoclonal antibody for tropane alkaloids (TAs) was established for the rapid screening of atropine, scopolamine, homatropine, apoatropine, anisodamine, anisodine and L-hyoscyamine residues in pig urine, pork and cereal flour samples through a simple sample preparation procedure. The half inhibitory concentrations of atropine, homatropine, L-hyoscyamine, apoatropine, scopolamine, anisodamine and anisodine were 0.05, 0.07, 0.14, 0.14, 0.24, 5.30 and 10.15 ng mL-1, respectivelyThe detection and quantitative limits of this method for TAs in samples were 0.18-73.18 and 0.44-74.77 µg kg-1. The spiked recoveries ranged from 69.88% to 147.93%, and the coefficient of variations were less than 14%. Good correlation (R2 = 0.9929) between the results of the ic-ELISA and the high performance liquid chromatography-tandem mass spectrometry support the reliability of the developed ic-ELISA method.

[Analytical investigation of a suspected case of drug-facilitated crime]. / Säker och känslig analysmetodik viktig vid misstänkta fall av ofrivillig drogning.

Sometimes it is suspected that people have been involuntary exposed to drugs, usually by spiked drinks. A young woman was transported to an emergency department by ambulance. Her clinical symptoms (decreased consciousness, mydriasis, confusion, hallucinations and urine retention) indicated anticholinergic syndrome that was effectively treated with the antidote physostigmine. A urine sample tested negative for common narcotic drugs and alcohol, but an extended toxicological analysis of the urine revealed the presence of the alkaloid scopolamine. Scopolamine occurs naturally in Solanaceae plants and is used in some medications. The woman reported that the symptoms had appeared soon after she was offered tea by a male acquaintance. The analytical results along with the woman's story indicated that she had been subjected to a drug-facilitated crime. The results further demonstrate that in suspected cases of involuntary drug exposure, testing should cover a wide panel of relevant drugs, otherwise poisoning may be missed.

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of in vivo and in vitro metabolites of scopolamine in rats.

In vivo and in vitro metabolism of scopolamine is investigated using a highly specific and sensitive liquid chromatography-mass spectrometry (LC-MSn) method. Feces, urine, and plasma samples are collected individually after ingestion of 55 mg/kg scopolamine by healthy rats. Rat feces and urine samples are cleaned up by a liquid-liquid extraction and a solid-phase extraction procedure (C18 cartridges), respectively. Methanol is added to rat plasma samples to precipitate plasma proteins. Scopolamine is incubated with homogenized liver and intestinal flora of rats in vitro, respectively. The metabolites in the incubating solution are extracted with ethyl acetate. Then these pretreated samples are injected into a reversed-phase C18 column with mobile phase of methanol-ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected by an on-line MSn system. Identification and structural elucidation of the metabolites are performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MSn spectra with those of the parent drug. The results reveal that at least 8 metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, hydroxyscopolamine, and hydroxyscopolamine N-oxide) and the parent drug exist in feces after administering 55 mg/kg scopolamine to healthy rats. Three new metabolites (tetrahydroxyscopolamine, trihydroxy-methoxyscopolamine, and dihydroxy-dimethoxyscopolamine) are identified in rat urine. Seven metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, and hydroxyscopolamine) and the parent drug are detected in rat plasma. Only 1 hydrolyzed metabolite (scopine) is found in the rat intestinal flora incubation mixture, and 2 metabolites (aposcopolamine and norscopolamine) are identified in the homogenized liver incubation mixture.

Confirmed Datura poisoning in a horse most probably due to D. ferox in contaminated tef hay.

Two out of a group of 23 mares exposed to tef hay contaminated with Datura ferox (and possibly D. stramonium) developed colic. The 1st animal was unresponsive to conservative treatment, underwent surgery for severe intestinal atony and had to be euthanased. The 2nd was less seriously affected, responded well to analgesics and made an uneventful recovery. This horse exhibited marked mydriasis on the first 2 days of being poisoned and showed protracted, milder mydriasis for a further 7 days. Scopolamine was chemically confirmed in urine from this horse for 3 days following the colic attack, while atropine could just be detected for 2 days. Scopolamine was also the main tropane alkaloid found in the contaminating plant material, confirming that this had most probably been a case of D. ferox poisoning. Although Datura intoxication of horses from contaminated hay was suspected previously, this is the 1st case where the intoxication could be confirmed by urine analysis for tropane alkaloids. Extraction and detection methods for atropine and scopolamine in urine are described employing enzymatic hydrolysis followed by liquid-liquid extraction and liquid chromatography tandem mass spectrometry (LC/MS/MS).

[Toxicological analysis of a case of Datura stramonium poisoning].

We encountered a patient in a restless excitable state after eating boiled jimson weed grown in the patient's garden. The patient mistook the weed for Angelica keiskei. Pupillary dilation (7/7mm), weak light reflex, body temperature of 37 degrees C, respiratory frequency of 19/min, blood pressure of 138/88 mmHg, pulse rate of 108/min, and hot feeling were observed. No abnormalities nor special findings were detected by general examination of the peripheral blood, biochemical examination of the blood, general examination of the urine, or electrocardiography. Atropine and scopolamine, which are tropane alkaloids, were detected by the GC/MS. The retention time of atropine-TMS was 17.0 min, and the mass spectra were m/z 124, 82, and 140. The retention time of scopolamine-TMS was 17.7 min, and the mass spectra were m/z 138, 108, 154 and 375. At the time of consultation, the serum concentrations of atropine and scopolamine were 31.3 ng/ml, and 30.6 ng/ml, respectively, and decreased to 6.7 ng/ml and 8.5 ng/ml, respectively, after 2 hours. The patient underwent injection of activated carbon after gastrolavage with 2,000 ml warm water, and neostigmine was administered. The patient awoke the following morning, and was discharged with mild pupillary dilation 2 days after poisoning.

Dose escalation pharmacokinetics of intranasal scopolamine gel formulation.

Astronauts experience Space Motion Sickness requiring treatment with an anti-motion sickness medication, scopolamine during space missions. Bioavailability after oral administration of scopolamine is low and variable, and absorption form transdermal patch is slow and prolonged. Intranasal administration achieves faster absorption and higher bioavailability of drugs that are subject to extrahepatic, first pass metabolism after oral dosing. We examined pharmacokinetics of 0.1, 0.2, and 0.4 mg doses of the Investigational New Drug formulation of intranasal scopolamine gel (INSCOP) in 12 healthy subjects using a randomized, double-blind cross-over study design. Subjects received one squirt of 0.1 g of gel containing either 0.1 mg or 0.2 mg/0.1 mL scopolamine or placebo in each nostril. Serial blood samples and total urine voids were collected after dosing and drug concentrations were determined using a modified LC-MS-MS method. Results indicate dose-linear pharmacokinetics of scopolamine with linear increases in Cmax and AUC within the dose range tested. Plasma drug concentrations were significantly lower in females than in males after administration of 0.4 dose. All three doses were well tolerated with no unexpected or serious adverse side effects reported. These results suggest that intranasal scopolamine gel formulation (INSCOP) offers a fast, reliable, and safe alternative for the treatment of motion sickness.

Transdermal hyoscine (Scopolamine). A preliminary review of its pharmacodynamic properties and therapeutic efficacy.

Hyoscine (scopolamine) is a competitive inhibitor of the muscarinic receptors of acetylcholine and it has been shown to be one of the most effective agents for preventing motion sickness. However, a relatively high incidence of side effects and a short duration of action has restricted the usefulness of this agent when administered orally or parenterally, and to counter this a novel transdermal preparation of hyoscine has been developed. Pharmacokinetic studies indicate that this new method for administering hyoscine controls the absorption process and the rate of drug entry into the systemic circulation over an extended period (72 hours), providing a means of delivery which is similar to a slow intravenous infusion. However, recent evidence suggests that the response to transdermal hyoscine treatment is variable and this may reflect pharmacokinetic differences between individuals. Controlled therapeutic trials have indicated that a single transdermal hyoscine patch is significantly superior to placebo and oral meclozine (meclizine) in preventing motion sickness. Trials comparing transdermal hyoscine with oral dimenhydrinate have failed to establish any significant differences in efficacy between the 2 drugs in small numbers of subjects, although there was always a more favourable trend towards the transdermal system. In patients with acute vertigo, transdermal hyoscine and oral meclozine were equally efficacious and both were significantly better than placebo in reducing the number of attacks of vertigo. Although transdermal hyoscine has been associated with a lower incidence of side effects than orally or parenterally administered hyoscine hydrobromide, adverse systemic effects have still been frequently reported. Most commonly cited have been dry mouth, drowsiness and impairment of ocular accommodation, including blurred vision and mydriasis (some ocular effects reported may be due to finger-to-eye contamination). Adverse central nervous system (CNS) effects, difficulty in urinating, rashes and erythema have been reported only occasionally. Thus, preliminary evidence suggests transdermal hyoscine may offer an effective and conveniently administered alternative for the prevention of motion-induced nausea and vomiting in certain situations. However, the duration of its clinical effectiveness, and its relative efficacy and tolerability compared with other agents needs to be confirmed in a few additional well-designed studies.

Pharmacokinetic consequences of spaceflight.

Spaceflight induces a wide range of physiological and biochemical changes, including disruption of gastrointestinal (GI) function, fluid and electrolyte balance, circulatory dynamics, and organ blood flow, as well as hormonal and metabolic perturbations. Any of these changes can influence the pharmacokinetics and pharmacodynamics of in-flight medication. That spaceflight may alter bioavailability was proposed when drugs prescribed to alleviate space motion sickness (SMS) had little therapeutic effect. Characterization of the pharmacokinetic and/or pharmacodynamic behavior of operationally critical medications is crucial for their effective use in flight; as a first step, we sought to determine whether drugs administered in space actually reach the site of action at concentrations sufficient to elicit the therapeutic response.

Interaction of alcohol and transdermally administered scopolamine.

In a placebo-controlled, randomized, double-blind cross-over study in 12 healthy volunteers the effect of acute alcohol intake during treatment with transdermally administered scopolamine (TTS-scopolamine) was investigated. One group of six subjects reached maximal blood alcohol concentrations (BAC) of 80 mg/dL and another group of six subjects a BAC of 130 mg/dL. There was no significant potentiation of alcohol effects on critical flicker fusion frequency by TTS-scopolamine. Sensorimotor function (choice reaction task) was also not significantly more influence by the combination. There was no effect of scopolamine on the elimination of alcohol. The urinary excretion of scopolamine was not influenced by oral intake of alcohol. TTS-scopolamine caused only minor side effects in a few volunteers, such as dry mouth (2 of 12) and blurred vision (1 of 12).

Preparation of a molecularly imprinted polymer for the solid-phase extraction of scopolamine with hyoscyamine as a dummy template molecule.

Molecularly imprinted polymers (MIPs) selective for scopolamine were produced using hyoscyamine (a close structural analogue) as template molecule. The produced polymers were used as media for solid-phase extraction, exhibiting selective binding properties for the analyte from biological samples. Human and calf urine and serum were processed on the MIP under various extraction protocols. The best performance was observed after loading the analyte in aqueous environment facilitating retention on the MIP by non-selective hydrophobic interactions. The MIPs were subsequently washed using an optimised solvent system to enable selective desorption of the analyte. Other related and non-related compounds were accessed to evaluate molecular recognition properties. Recoveries of up to 79% were achieved for the analyte of interest from biological samples.

Submicrogram assay for scopolamine in plasma and urine.

A GLC-mass spectrometric method for scopolamine, sensitive to 50 pg/ml for a 4-ml plasma or urine sample, was developed. The method used a deuterated internal standard to minimize variability in absolute recovery in the extraction procedure. Scopoline and deuterated scopoline were formed from the base-catalyzed hydrolysis of scopolamine and the internal standard and were analyzed as the heptafluorobutyrates, using a GLC-mass spectrometric system by monitoring the m/e 138 and 141 fragments, respectively.

A sensitive radioreceptor assay for determining scopolamine concentrations in plasma and urine.

A sensitive and reliable procedure for the quantitation of low picogram levels of scopolamine in plasma and urine is described. The method consists of two steps, a preparative extraction step using C18 columns (Sep-Pak), followed by an analytical quantitation step involving a muscarinic radioreceptor assay. The extraction efficiency of the C18 columns was 85-95% for both plasma and urine over a wide concentration range. When [3H]methyl scopolamine is used as a tracer, the assay can detect picogram concentrations (greater than 25 pg) of scopolamine (base) in plasma and urine. The applicability of the procedure for therapeutic drug monitoring of scopolamine was demonstrated by using the method to determine plasma levels in humans after transdermal administration.

Simple radioligand binding assay for the determination of urinary scopolamine.

A sensitive radioligand binding assay is described for the determination of scopolamine in human urine. As a measure for the drug concentration, the quantitative displacement of scopolamine of tritiated quinuclidinyl benzylate from rat brain receptors was used. The assay is sensitive to concentrations as low as 1.2 ng/mL, surpassed only by GC-MS techniques. It can be performed easily and quickly and does not include extraction procedures. Scopoline and scopine , possible metabolites of scopolamine, do not interfere with the assay. After transdermal administration of scopolamine, 34% of the drug is found in the urine. Of the total scopolamine excreted, 79% is conjugated to glucuronic and/or sulfuric acid and 21% is excreted in the unbound form.

Transdermally administered scopolamine vs. dimenhydrinate. I. Effect on nausea and vertigo in experimentally induced motion sickness.

The effect of transdermally administered scopolamine (TTS-scopolamine) (2.5 cm2 surface area, one and two patches) and dimenhydrinate (100 mg) on experimental motion sickness was examined in 16 healthy volunteers in a randomized double-blind study. Nausea was induced by Coriolis manoeuvre and vertigo by calorization of the ear. In all subjects, scopolamine was found in urine in concentrations indicating adequate absorption of the drug. One TTS-scopolamine, two TTS-scopolamine and dimenhydrinate caused a statistically significant reduction in nausea when compared with placebo. Dimenhydrinate was somewhat more effective against nausea than one TTS-scopolamine. Vertigo was significantly reduced after dimenhydrinate and two TTS-scopolamine. Side effects of both drugs were negligible, though gait disturbances and vertigo could occur occasionally after two TTS-scopolamine. No dose-response relationship was found between the urinary excretion of scopolamine and alleviation of nausea. Dimenhydrinate and TTS-scopolamine are both effective against motion sickness, the latter provided it is applied 6 to 8 hours before exposure to the stimulus causing the motion sickness.

Determination of drugs used as anti-Parkinson's disease drugs in urine and serum by capillary electrophoresis.

A new capillary electrophoresis method to determine simultaneously eight of the most important anti-Parkinson's disease compounds has been developed. The generic names of the drugs studied are benactyzine (BA), trihexyphenidyl (TP), fenpiverin (FP), diphemin (DF), scopolamine (BL), adiphenine (TS), diethylaminoethylester 1-phenylcyclopentane-1-carboxylate (EKK), and diethylaminoethylester tetramethoxydiphenylacetate (EKO). An untreated fused-silica capillary tube (75 microns i.d., 57 cm total length, 49.5 cm length to the detector) was used with detection at 190 nm. The optimal separation conditions were 50 mM phosphate buffer (pH 2.7) with 7 mM-beta-cyclodextrin, electrokinetic injection for 15 sec at 5 kV, temperature 25 degrees C, and 15-20 kV separation voltage. Complete separation of all compounds was achieved in less than 16 min. The procedure was applied for the determination in urine and serum. The limits of detection (LOD, S/N = 3) for serum were 209 (FP), 234 (EKO), 168 (DF), 182 (BA), 168 (TP), 220 (BL), 174 (TS), and 163 (EKK) ppb. The method can be used for the therapeutic drug monitoring of these central active cholinolytics in clinical laboratories.