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.

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.

[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.

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.

[Determination of hyoscyamine and scopolamine in serum and urine of humans by liquid chromatography with tandem mass spectrometry].

A simple method was developed for the analysis of hyoscyamine and scopolamine in human serum and urine using liquid chromatography with tandem mass spectrometry (LC/MS/MS). Hyoscyamine and scopolamine in serum and urine were cleaned up with an Oasis HLB cartridge and a PSA cartridge. The LC separation was carried out on an ODS column, using linear gradient elution with 5 mmol/L IPCC-MS3-methanol as the mobile phase. The mass spectral acquisition was done in the positive ion mode by applying selected reaction monitoring (SRM). The recoveries of hyoscyamine and scopolamine were 86.0-105% from human serum and urine fortified at 0.2 ng/mL and 10 ng/mL. The detection limits of hyoscyamine and scopolamine were 0.02 ng/mL. Four serum and three urine samples of humans poisoned by eating Datura innoxia Mill. were analyzed by this method. Hyoscyamine and scopolamine were detected at the levels of 0.45-3.5 ng/mL in all serum samples and 170-670 ng/mL in all urine samples.

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.

Application of high performance capillary electrophoresis on toxic alkaloids analysis.

We employed CE to identify mixtures of the toxic alkaloids lappaconitine, bullatine A, atropine sulfate, atropine methobromide, scopolamine hydrobromide, anisodamine hydrobromide, brucine, strychnine, quinine sulfate, and chloroquine in human blood and urine, using procaine hydrochloride as an internal standard. The separation employed a fused-silica capillary of 75 microm id x 60 cm length (effective length: 50.2 cm) and a buffer containing 100 mM phosphate and 5% ACN (pH 4.0). The sample was injected in a pressure mode and the separation was performed at a voltage of 16 kV and a temperature of 25 degrees C. The compounds were detected by UV absorbance at wavelengths of 195 and 235 nm. All the ten alkaloids were separated within 16 min. The method was validated with regard to precision (RSD), accuracy, sensitivity, linear range, LOD, and LOQ. In blood and urine samples, the detection limits were 5-40 ng/mL and linear calibration curves were obtained over the range of 0.02-10 microg/mL. The precision of intra- and interday measurements was less than 15%. Electrophoretic peaks could be identified either by the relative migration time or by their UV spectrum.

[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.

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).

Urinary excretion of dietary contaminants in horses.

REASONS FOR PERFORMING STUDY: Presence of drugs is completely prohibited in post racing urine samples by most of racing and competition authorities, even if environmental contamination might occur. OBJECTIVES: To assess the daily dose of several contaminants absorbed through the diet that would result in detectable concentrations in urine. METHODS: Caffeine, theobromine, theophylline, atropine, scopolamine, bufotenine, DMT or morphine were administered orally to 6 horses, in different dosages, for 3 days before their urine was sampled for regular anti-doping tests. RESULTS: Theobromine, theophylline, bufotenine and morphine daily intake >10 mg, 2 mg, 10 mg and 200 microg, respectively, by a performance horse, were found to result in detectable urinary concentrations. At the 2 tested doses, atropine (5 and 15 mg) and dimethyltryptamine (3 and 10 mg) were not detected in urine. For caffeine and scopolamine, even the lowest dosage tested (5 mg/horse/day and 2 mg/horse/day respectively) induced detectable concentrations of the molecule in urine. CONCLUSIONS: Horses fed dietary contaminants, even at level much below the effective dosage, may be positive to antidoping urine analysis. Further research is needed to gain more confident results on a daily safe intake for caffeine and scopolamine. POTENTIAL RELEVANCE: Selection of feed materials appears to be of great importance to prevent non voluntary positive result to anti-doping tests.

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.

Effects of antimuscarinic drugs on both urinary frequency and cognitive impairment in conscious, nonrestrained rats.

Recent studies indicate a risk of learning and memory impairments when patients with senile dementia are treated with antimuscarinic drugs. In this study, we compared the effectiveness of propiverine hydrochloride (propiverine) and oxybutynin chloride (oxybutynin) on the increased urinary frequency and cognitive impairment induced by nucleus basalis magnocellularis (nBM) lesioning in conscious and nonrestrained rats. For examination of bladder function, nBM-lesioned rats were given total parenteral nutrition regimens for 8 days. Propiverine administered orally at 0.3, 3 and 30 mg/kg on the postoperative day 7 significantly lessened the increase in the frequency of voiding caused by the nBM lesion, whereas oxybutynin administration did not show any improvement at 0.1 or 1 mg/kg but did so at 10 mg/kg. To examine the memory impairment, we trained nBM-lesioned rats in an 8-arm radial maze task for 20 days and then evaluated the effectiveness of oral drug administration on 19th and 20th radial maze performance. The higher rate of errors caused by nBM lesioning was significantly aggravated by oxybutynin at 30 and 100 mg/kg. Propiverine showed slight aggravation of errors, but with no statistical significance at any dose, 30, 100 or 300 mg/kg. These results suggest that propiverine has comparatively less effect on the cognitive impairment than oxybutynin.

Laboratory confirmation of scopolamine co-intoxication in patients using tainted heroin.

BACKGROUND: First described in 1995, at least 325 patients with a history of heroin use have since required emergency medical evaluation in several eastern US cities, because of an anticholinergic toxidrome following use of heroin. This co-intoxication has been alleged to result from an atropine-like compound. We report the clinical findings and laboratory analysis of one of several individuals who presented to our Emergency Department during this epidemic. CASE REPORT: A 23-year-old male was one of 3 patients brought to the Emergency Department due to agitated behavior after insufflating heroin. Following physical and chemical restraint, vital signs were pulse 134 bpm, BP 160/90 mm Hg, RR 24/min, and T 37.3 degrees C. Physical examination was remarkable for dilated pupils 8-9 mm without nystagmus, along with dry mouth, decreased bowel sounds, and flushed dry skin. A bladder catheter was placed and 500 mL of urine was obtained. Electrocardiogram revealed sinus tachycardia only. Additional sedation was required for 12 hours until normal mental status returned. A small sample of the "heroin" used was obtained and analyzed. Scopolamine was confirmed by gas chromatography-mass spectrometry. Further evidence of scopolamine intoxication was supported by identifying scopolamine in the urine of this patient. CONCLUSION: Gas chromatography-mass spectrometry revealed scopolamine to be the cause of anticholinergic findings in a patient following use of tainted heroin. It is unclear whether scopolamine is an adulterant or contaminant in this heroin. Patients with anticholinergic findings following use of heroin may be co-intoxicated with scopolamine.

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.

Liquid chromatographic determination of hyoscine (scopolamine) in urine using solid phase extraction.

A sensitive method for the determination of hyoscine (scopolamine) in urine is described. After concentration and "clean-up" on C18 and CN solid phase extraction columns, hyoscine was quantified by high performance liquid chromatography with coulometric detection (oxidation at +0.9 V). The limit of detection was 5 ng per sample and the precision for 5 mL samples containing 2 ng/mL was 12.3%. The method was applied to urine samples collected from 12 volunteers wearing Scopoderm TTS patches. The mean excretion rate of unmetabolized hyoscine was 0.45 micrograms/h and 87% of the total hyoscine was present as conjugates. Apohyoscine (aposcopolamine) was identified as a urinary metabolite. The significance of this with regard to hyoscine assays is discussed.

Metabolism in vivo of the tropane alkaloid, scopolamine, in several mammalian species.

1. In vivo metabolism of scopolamine was studied in rats, mice, guinea pigs and rabbits. The structures of eight urinary metabolites including unchanged drug were elucidated by mass and nuclear magnetic resonance spectrometry. Determination of these metabolites was achieved by a g.l.c. method using a semi-capillary column. 2. The major metabolites in rats were the three phenolic metabolites, p-hydroxy-, m-hydroxy- and p-hydroxy-m-methoxy-scopolamine. 3. Significant intra-species difference of the metabolism was observed in rabbits. Tropic acid was the major metabolite in two rabbits out of three, while the other rabbit excreted mainly unchanged scopolamine, accompanied by five metabolites. Tropic acid was also the major metabolite in guinea pigs, but was of minor importance in mice. 4. The dehydrated metabolites, aposcopolamine and aponorscopolamine, were abundantly excreted in guinea pigs, moderately in mice, and least in rabbits and rats. 5. Excretion of glucuronide conjugates of scopolamine and norscopolamine were high in mice compared with other species. On the other hand, phenolic metabolites in rat urine; and tropic acid in rabbit and guinea pig urine, were excreted as the free forms. 6. These results indicate that scopolamine metabolism is highly species-specific.

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.

beta-Glucuronide and sulfate conjugation of scopolamine and glycopyrrolate.

The metabolism of scopolamine and glycopyrrolate was studied in 11 healthy parturients undergoing cesarean section. After a single intramuscular injection of scopolamine (5 micrograms/kg, n = 7) or glycopyrrolate (6 micrograms/kg, n = 4), the concentrations of the drugs in the urine were determined up to 8-12 h using a radioreceptor assay. This assay measures scopolamine and glycopyrrolate with their possible active metabolites. The effect of beta-glucuronidase and sulfatase incubation on the drug concentrations was also studied. The concentrations of scopolamine and/or its active metabolites were on the average 7 times higher after incubation indicating that beta-glucuronide or sulfate conjugation is an important metabolic pathway for scopolamine. On the contrary, the glycopyrrolate concentrations increased only slightly between 1 and 3 hours after the drug injection. Thus, beta-glucuronide or sulfate conjugation plays only a minor part in the metabolism of glycopyrrolate.

Pharmacokinetics of scopolamine during caesarean section: relationship between serum concentration and effect.

The pharmacokinetics (radioreceptor assay, RRA) and some of the clinical effects of the anticholinergic agent, scopolamine, were studied in 16 parturients during caesarean section. Following a single 0.005 mg/kg intramuscular injection (deltoid muscle), a very fast rate of absorption was found with mean peak serum concentrations occurring after only 10 min (n = 6). Due to severe bradycardia, 0.5 mg of atropine i.v. had to be given in addition to the i.m. scopolamine treatment to one parturient. The RRA measured the total concentration produced by the two anticholinergic agents in both serum and urine. There was a fundamental difference in the diffusion of scopolamine through the placenta and the blood-lumbar (CSF-barrier (n = 15). There was significant drug penetration in the foeto-placental unit, indicating an efficient drug transfer to the child, but there were measurable levels of the drug in the lumbar CSF in only three cases. The apparent elimination phase half-life of scopolamine in serum was only around 1 h. The urinary excretion of scopolamine and/or its antimuscarinic metabolites lasted only for 6 h (2.63 +/- 1.14% of the dose). The onset of the clinical effects of scopolamine appeared to be delayed, but long-lasting in contrast to the rapid absorption and quick disappearance from the serum. Both the heart rate changes, sedative and antisialogogue effects and serum concentrations did not show any correlation. There appears to be a surprisingly great difference between the pharmacokinetic parameters and the clinical effects of scopolamine.