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.

Preventive Effects of Dexmedetomidine on Renal Dysfunction and Hemodynamic Stability in Malignant Obstructive Jaundice Patients During Peri-Operative Period.

BACKGROUND This study aimed to investigate effects of intra-operative administration with dexmedetomidine (Dex) on hemodynamics and renal function in patients with malignant obstructive jaundice. MATERIAL AND METHODS Our randomized, double-blinded, placebo-controlled study was conducted among 40 patients with malignant obstructive jaundice between August 2009 and March 2011 in The Affiliated Hospital of Inner Mongolia Medical University. The 40 patients were randomly divided into 2 groups: the Dex group (receiving Dex 0.5 µg/kg 10-minutes before induction and then a 0.5 µg/kg/hour maintenance infusion until end of operation 30 minutes) and the Control group (receiving normal saline of same amount and at same rate). The adverse events, including incidence of cardiovascular complications and nausea and vomiting, and length of hospital stay were determined. The level of cystatin C (CysC), retinol-binding protein (RBP), creatinine (Scr), and blood urea nitrogen (BUN) were also evaluated. RESULTS Dexmedetomidine administration significantly decreased heart rate (HR) and stroke volume variation (SVV) and significantly increased capital venous pressure (CVP) and mean arterial pressure (MAP) values compared to that in the Control group (P<0.05). Dexmedetomidine administration significantly upregulated urine volume and significantly downregulated atropine levels compared to the Control group (P<0.05). Dexmedetomidine administration significantly improved renal functions, by modulating CysC, RBP, Scr and BUN levels compared to the Control group (P<0.05). Dexmedetomidine administration demonstrated no additional side-effects. Dexmedetomidine administration significantly shortened length of hospitalization in the Dex group compared to the Control group (P<0.05). CONCLUSIONS Dexmedetomidine plays preventive effects on renal dysfunction and hemodynamic stability in malignant obstructive jaundice patients during peri-operative period.

Highly sensitive determination of atropine using cobalt oxide nanostructures: Influence of functional groups on the signal sensitivity.

This study describes sensitive determination of atropine using glassy carbon electrodes (GCE) modified with Co3O4 nanostructures. The as-synthesised nanostructures were grown using cysteine (CYS), glutathione (GSH) and histidine (HYS) as effective templates under hydrothermal action. The obtained morphologies revealed interesting structural features, including both cavity-based and flower-shaped structures. The as-synthesised morphologies were noted to actively participate in electro-catalysis of atropine (AT) drug where GSH-assisted structures exhibited the best signal response in terms of current density and over-potential value. The study also discusses the influence of functional groups on the signal sensitivity of atropine electro-oxidation. The functionalisation was carried with the amino acids originally used as effective templates for the growth of Co3O4 nanostructures. The highest increment was obtained when GSH was used as the surface functionalising agent. The GSH-functionalised Co3O4-modified electrode was utilised for the electro-chemical sensing of AT in a concentration range of 0.01-0.46 µM. The developed sensor exhibited excellent working linearity (R2 = 0.999) and signal sensitivity up to 0.001 µM of AT. The noted high sensitivity of the sensor is associated with the synergy of superb surface architectures and favourable interaction facilitating the electron transfer kinetics for the electro-catalytic oxidation of AT. Significantly, the developed sensor demonstrated excellent working capability when used for AT detection in human urine samples with strong anti-interference potential against common co-existing species, such as glucose, fructose, cysteine, uric acid, dopamine and ascorbic acid.

Determination of atropine sulfate using a novel sensitive DNA-biosensor based on its interaction on a modified pencil graphite electrode.

A novel, selective, rapid and simple electrochemical method is developed for the determination of atropine sulfate. UV-Vis and differential pulse voltammetry are used to study the interaction of atropine sulfate with salmon sperm ds-DNA on the surface of salmon sperm ds-DNA modified-pencil graphite electrode (PGE). For this purpose, a pencil graphite electrode (PGE) modified with multiwall carbon nanotubes (MWCNTs), titanium dioxide nanoparticles (TiO2NPs), and poly-dialyldimethylammonium chloride (PDDA) decorated with ds-DNA is tested for the determination of atropine sulfate. The electrochemical oxidation peak current of adenine and guanine bonded on the surface of ds-DNA/PDDA-TiO2NPs-MWCNTs/PGE is used to obtain the analytical signal. Decreases in the intensities of guanine and adenine oxidation signals after their interaction with atropine sulfate are used as indicator signals for the sensitive determination of atropine sulfate. Using ds-DNA/PDDA-TiO2NPs-MWCNTs/PGE and based on the guanine signal, linear calibration curves were obtained in the range of 0.6 to 30.0 µmol L(-1) and 30.0 to 600.0 µmol L(-1) atropine sulfate with low detection limits of 30.0 nmol L(-1). The biosensor shows a good selectivity for the determination of atropine sulfate. Finally, the applicability of the biosensor is evaluated by measuring atropine sulfate in real samples with good accuracy.

Molecularly imprinted photonic hydrogels for fast screening of atropine in biological samples with high sensitivity.

Based on molecularly imprinted photonic hydrogels (MIPHs) that combined the colloidal-crystal with molecular imprinting technique, a novel label-free colorimetric chemosensor for convenient and fast efficient detection of atropine with high sensitivity and specificity was developed. Due to the special inverse opal arrays with a thin polymer wall in which the imprinted nanocavities of atropine moleculars distributed, the proposed MIPHs designed as water-compatible exhibited high sensitive (as low as 1 pg/mL), rapid responsive (less than 30 s) and specific detection of atropine in complex matrix. The unique three-dimensional, highly-ordered photonic hydrogels would be obviously swelling in response to the specific atropine molecular recognition process and the response would be directly transferred into visually perceptible optical signal (change in color) that could be detected by the naked eye through Bragg diffractive shifts of ordered macroporous arrays. With a broad concentration range varying from 1 pg/mL to 1 µg/mL of atropine, the distinct color changes of MIPHs almost covered the whole visible-light wavelength range from blue to red for semi-quantitative analysis. The smart chemosensor was successfully employed to determine the trace level atropine in human urine samples, providing a fast and effective alternative for semi-quantitative detection of atropine for clinical analysis and forensic investigations.

[Detection of blood atropine by gas chromatography with a mass-selective detector].

A method for the detection of atropine in cadaveric blood has been developed using a gas chromatograph equipped with a mass-selective detector. The concentration range of the method is 20-1000 ng/ml, the limit of sensitivity 10 ng/ml. Maximum intra-assay error does not exceed 12% and 6% for concentrations of 20 and 300 ng/ml respectively. The method was validated using material for forensic chemical examination.

[Determinations of the presence of drugs in traffic users in the material of the Department of Forensic Medicine, Medical University of Bialystok]. / Badania nad obecnoscia srodków odurzajacych i substancji psychotropowych w organizmach uczestników ruchu drogowego w materiale Zakladu Medycyny Sadowej UM w Bialymstoku.

In recent years, there has been observed an increasing number of traffic users being under influence of psychoactive substances that affect the central nervous system. A total of 198 blood samples and 23 urine samples collected from traffic users (drivers, passengers and pedestrians) suspected of having ingested psychoactive substances were examined. The analysis included blood samples collected from living individuals and blood or urine from the deceased. Ethyl alcohol levels were determined by gas chromatography, while body fluids were examined by Elisa tests for determination of cannabinoids, amphetamines, opium narcotics, cocaine (benzoiloecgonine), benzodiazepines, barbiturates and tricyclic antidepressants. The confirmation of positive results was carried out by gas chromatography with mass detector. Twenty-nine blood samples were positive, what constituted 14.6% of the total number of investigated cases, including 12 (7.8%) of samples originating from living individuals and 17 (37.8)--from the fatalities. In both groups, the most commonly detected substances were cannabinoids (THC and its metabolite carboxy-THC) and amphetamines and its analogues.

[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.

Pitfalls and prevention strategies for liquid chromatography-tandem mass spectrometry in the selected reaction-monitoring mode for drug analysis.

BACKGROUND: We observed cases of false-positive results with the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Different LC-MS/MS techniques that use the selected reaction-monitoring mode, routinely employed for the analysis and quantification of drugs and toxic compounds in biological matrices, were involved in the false-positive and potentially false-positive results obtained. We sought to analyze the causes of and solutions to this problem. METHODS: We used a previously reported LC-MS/MS general unknown screening method, as well as manual spectral investigation in 1 case, to perform verification and identification of interfering compounds. RESULTS: We observed that false-positive results involved: a metabolite of zolpidem that might have been mistaken for lysergic acid diethylamide, benzoylecgonine mistaken for atropine, and clomipramine and 3 phenothiazines that share several common ion transitions. CONCLUSIONS: To prevent problems such as those we experienced, we recommend the use of stable-isotope internal standards when possible, relative retention times, 2 transitions or more per compound when possible, and acceptable relative abundance ratios between transitions, with an experience-based tolerance of +/-15% for transitions with a relative abundance >10% and with an extension to +/-25% for transitions <10% when the concentration is at the limit of quantification. A powerful general unknown screening procedure can help to confirm suspected interferences. Our results indicate that the specificity of screening procedures is questionable for LC-MS/MS analyses performed in the selected reaction-monitoring mode and involving a large number of compounds with only 1 transition per compound.

[Determination of fentanyl, atropine and scopolamine in biological material using LC-MS/APCI methods]. / Oznaczanie fentanylu, atropiny i skopolaminy w materiale biologicznym metodami LC-MS/APCI.

In this paper methods for determination of fentanyl (FL) and its three analogues: alfentanyl (AL), sufentanyl (SL) and remifentanyl (RL), atropine (AT) and scopolamine (SK) in biological material (whole blood and urine) using liquid chromatography atmospheric pressure chemical ionisation mass spectrometry technique (LC-MS/ APCI) are presented. Separation of analytes was performed in gradient conditions, using a LiChroCART LiChrospher 60 RP-select B column. The mobile phase consisted of 0.1% (v/v) formic acid in water and in acetonitrile. Target analytes were isolated from biological matrices using liquid-liquid extraction technique with n-butyl chloride or diethyl ether as extraction solvents. The validation data of the methods were: limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) - 0.05 and 0.25 ng/ml for FL, and 0.7 and 0.9 ng/ml for AT, both in blood, whereas 1.9 and 2.1 ng/ ml for FL, and 0.6 and 0.9 ng/ml for AT in urine. Calibration curves showed linearity in concentration ranges from LOQ to 25 ng/ml in blood and from LOQ to 50 ng/ml in urine. Determination coefficients (R2) of linear regression equation were higher then 0.98. Extraction recovery, intra-day precision (CV(w.g.)) and inter-day precision (CV(m.g.)) were determined at analytes and internal standard (I.S.) concentration of 5 ng/ml for blood, and at analytes and I.S. concentrations of 20 and 5 ng/ml, respectively for urine. Extraction recovery ranged from 76 to 100% for blood and 53--72% for urine. CV (n=5) and CV(m.g.) (n=15) equal from 4.8 to 7.5% and from 6.8 to 16.2% respectively for blood, and from 4.3 to 5.4% and from 5.8 to 9.5% for urine. The application of elaborated methods of the determination of FL, AT and SK in blood and urine for 8 expert opinions elaborated at the Institute of Forensic Research in Krakow is described. FL was detected and quantified in 3 cases, whereas AT and SK in 7.

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.

[Acute Datura stramonium poisoning in an emergency department]. / Intoxications aiguës à Datura stramonium aux urgences.

BACKGROUND: The toxic effects of Datura stramonium most often include visual and auditory hallucinations, confusion and agitation. Severe and even fatal complications (coma, respiratory distress or death in more than 5% of cases) are not rare since the lethal concentration of the drug's toxic substances (i.e., atropine and scopolamine) is close to the level at which delirium occurs. CASES: A 17-year-old man was admitted to our emergency department with agitation, delirium with persecutory ideation and frightening hallucinations of being assaulted by animals. Blood samples taken 12 hours after Datura stramonium ingestion and analyzed with liquid chromatography and mass spectrometry (LC-MS/MS) found 1.7 ng/mL of atropine, close to the lethal level. After restraint and treatment with the antipsychotic drug cyamemazine, the young man returned to normal 36 hours after drug ingestion. A 17-year-old woman was admitted to our emergency department after losing consciousness on a public thoroughfare. At the emergency department, 2 hours after she had ingested Datura stramonium, she was agitated, with delirium, anxiety, and frightening visual and tactile hallucination of green turtles walking on her as well as auditory hallucinations. Blood samples at D0, D1 and D2 after Datura stramonium ingestion, analyzed with LC-MS/MS, found: 1.4, 1.0, and 0.2 ng/mL of scopolamine, respectively. Atropine was massively eliminated in urine on D1 (114 ng/mL). After restraint and cyamemazine treatment, the young woman returned to normal 40 hours after she had first ingested this hallucinogen. DISCUSSION: These cases of intoxication with Datura stramonium are, to our knowledge, the first clinical reports correlated with toxicologic analysis by the reference method (LC-MS/MS) in an emergency setting. Since neither the drug-users nor those accompanying them usually volunteer information about drug use, it is important to consider this specific risk in cases of agitation and confusion in adolescents or young adults.

[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).

Sensitive and specific liquid chromatographic-tandem mass spectrometric assay for atropine and its eleven metabolites in rat urine.

A sensitive and specific method is described for the simultaneous determination of atropine and its metabolites in rat urine by combining liquid chromatography and tandem mass spectrometry (LC-MS(n)). Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of atropine. After extraction procedure the pretreated samples were separated on a reversed-phase C18 column using a 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 LC-MS(n) system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. The results revealed that at least eleven metabolites (N-demethyltropine, tropine, N-demethylatropine, p-hydroxyatropine, p-hydroxyatropine N-oxide, glucuronide conjugates and sulfate conjugates of N-demethylatropine, p-hydroxyatropine and the parent drug) and the parent drug existed in rat urine after ingesting 25 mg/kg atropine. p-Hydroxyatropine and the parent drug were detected in rat urine for up 106 h after ingestion of atropine.

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.

[Psychoactive substances in biological samples--toxicological laboratory data]. / Substancje psychoaktywne w materiale biologicznym--dane z laboratorium toksykologicznego.

The subject of the research was the analysis of frequency and type of psychoactive substances used, basing on the determinations the blood and/or urine samples, performed in the toxicological laboratory of the Department of Clinical and Industrial Toxicology Jagiellonian University in Kraków in the period from December 2001 to November 2003. From 17,649 performed determinations--45.5% were positive. 50% of the positive determinations were psychoactive substances. The most often psychoactive substance determined was ethyl alcohol (52.86%), next benzodiazepines (17.41%), amphetamines (10.54%), opiates (8.05%), THC (6.87%), barbiturates (3.74%), and occasionally atropine and cocaine. There was observed a variety of mixed, simultaneously taking psychoactive substances, especially ethyl alcohol, opiates, amphetamine derivatives and cannabinoids. The analysis of the occurrence of psychoactive substances in biological samples from patients treated in different hospital departments, others hospitals and ordered by private persons also was performed. In the last two years 369 private patients ordered psychoactive substances determinations and 78 of them were positive.

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.

Tris(2,2'-bipyridine)ruthenium(II) electrogenerated chemiluminescence of alkaloid type drugs with solid phase extraction sample preparation.

An electrogenerated chemiluminescence (ECL) method for the determination of pethidine, atropine, homatropine and cocaine is described. The optimum conditions were found to be similar for all of these compounds although the ECL emission intensity for cocaine was an order of magnitude lower than for pethidine due to their different chemical structures. Linear calibrations were obtained for all the compounds at pH 10 in borate buffer (0.05 mol l-1) at 1.3 V. Limits of detection of 6.8 x 10(-8), 2.2 x 10(-7), 3.2 x 10(-7) and 6.5 x 10(-7) mol l-1, respectively, were achieved for pethidine, atropine, homatropine and cocaine in standard solutions. Solid-phase extraction was used to separate the drugs from their matrix and the method was applied to the determination of spiked urine samples. The limits of quantitation for pethidine, atropine, homatropine and cocaine in urine were 1.0 x 10(-6), 2.0 x 10(-6), 2.0 x 10(-6) and 4.0 x 10(-6) mol l-1, respectively, with recoveries of between 90 and 110%.