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.

Calystegines are Potential Urine Biomarkers for Dietary Exposure to Potato Products.

SCOPE: Metabolites derived from specific foods present in urine samples can provide objective biomarkers of food intake (BFIs). This study investigated the possibility that calystegines (a class of iminosugars) may provide BIFs for potato (Solanum tuberosum L.) product exposure. METHODS AND RESULTS: Calystegine content is examined in published data covering a wide range of potato cultivars. Rapid methods are developed for the quantification of calystegines in cooked potato products and human urine using triple quadrupole mass spectrometry. The potential of calystegines as BFIs for potato consumption is assessed in a controlled food intervention study in the United Kingdom and validated in an epidemiological study in Portugal. Calystegine concentrations are reproducibly above the quantification limit in first morning void urines the day after potato consumption, showing a good dose-response relationship, particularly for calystegine A3 . The design of the controlled intervention mimicks exposure to a typical UK diet and showed that neither differences in preparation/cooking method or influence of other foods in the diet has significant impact on biomarker performance. Calystegine biomarkers also perform well in the independent validation study. CONCLUSION: It is concluded that calystegines have many of the characteristics needed to be considered as specific BFIs for potato product intake.

Phase I clinical study with different doses of 99mTc-TRODAT-1 in healthy adults.

OBJECTIVES: To study the pharmacokinetics, biodistribution, and injection doses of 99mTc-TRODAT-1 in healthy adults. METHODS: Thirty healthy individuals comprising 15 females and 15 males were randomly divided into three groups and the injection doses of 99mTc-TRODAT-1 of group 1, 2, and 3 were 370 MBq, 740 MBq, and 1110 MBq, respectively. Assessments of subjective symptoms and tests were performed before and after injection. Blood and urine collections and whole-body planar imaging were analyzed at various time points. Bilateral brain striatal SPECT images obtained at 3.5 h PI were assessed visually and semiquantitatively. RESULTS: No serious adverse events or deaths were observed in our study. The pharmacokinetic analysis showed that 99mTc-TRODAT-1 was eliminated rapidly from the circulation, with just about 4% of the injected dose remaining in blood at 1 h post-injection. The mean cumulative urinary excretion over 24 h was just 2.96 ± 0.96%ID. The time-activity curve demonstrated that the radioactivity was mainly in liver and abdomen. The highest absorbed dose was in the dose-limiting organ, liver (20.88 ± 4.45 × 10-3 mSv/MBq). The average effective dose was 5.22 ± 1.05 × 10-3 mSv/MBq. The clarity of striatal images assessed visually in group 1 was worse than that in group 2 and 3. The semiquantitative analysis showed that there were no differences in striatum/cerebellum between the three groups (group 1: 1.77 ± 0.11, group 2: 1.62 ± 0.14, and group 3: 1.75 ± 0.20; P = 0.088). CONCLUSIONS: 99mTc-TRODAT-1 was safe to use in humans and showed the status of dopaminergic neurons specifically and clearly. The injection dose we suggested was 740 MBq.

Mass balance and metabolism of aclidinium bromide following intravenous administration of [¹4C]-aclidinium bromide in healthy subjects.

Aclidinium bromide is a novel, inhaled long-acting muscarinic antagonist with low systemic activity developed for the treatment of COPD. It is an ester compound rapidly hydrolysed in plasma into inactive alcohol and acid metabolites. In this Phase I, open-label study, the rates and routes of elimination of radioactivity following intravenous administration of [¹4C]-aclidinium bromide were determined. The metabolites of aclidinium were also characterized and identified in plasma and excreta. Twelve healthy males were randomized (1:1) to receive a single intravenous 400 µg dose of [phenyl-U-¹4C]- or [glycolyl-U-¹4C]-aclidinium bromide (via 5 min infusion) to label alcohol or acid metabolites of aclidinium, respectively. Safety and tolerability were assessed over a 9-day period. Following intravenous administration, the parent compound was rapidly hydrolysed into its acid and alcohol metabolites. Primary excretion routes for [phenyl-U-¹4C]- and [glycolyl-U-¹4C]-aclidinium were renal (urine: 65% and 54%, respectively; feces: 33% and 20%, respectively), with 1% excreted as unchanged aclidinium. A total of three treatment-emergent adverse events in two subjects were reported and were related to infusion site pain. Overall, aclidinium is rapidly hydrolysed into two main metabolites, which are predominantly excreted in urine. Aclidinium bromide 400 µg administered intravenously was safe and well tolerated in healthy subjects.

Pharmacokinetics and safety of aclidinium bromide, a muscarinic antagonist, in adults with normal or impaired renal function: A phase I, open-label, single-dose clinical trial.

BACKGROUND: Aclidinium bromide is an inhaled, long-acting muscarinic antagonist currently in development for the treatment of chronic obstructive pulmonary disease. Renal impairment may affect drug clearance. OBJECTIVE: This study was conducted to evaluate the pharmacokinetic (PK) parameters, safety, and tolerability of aclidinium bromide and its metabolites in patients with normal and impaired renal function to determine whether dosing adjustments are required when renal dysfunction is present. METHODS: This was a Phase I, open-label, single-center, single-dose clinical trial conducted in Munich, Germany. Adults with varying degrees of renal function were assigned to 4 groups (n = 6 for each) based on creatinine clearance, including normal renal function (>80 mL/ min), mild renal insufficiency (>50-≤80 mL/min), moderate renal insufficiency (>30-≤50 mL/min), and severe renal insufficiency (<30 mL/min). Single doses of aclidinium bromide 400 µg were administered using a multidose dry powder inhaler. Blood and urine samples were obtained before dosing and at various time points up to 48 hours after dosing to analyze the PK parameters of aclidinium bromide and its metabolites. Plasma PK Parameters were AUC0₋(t), MJC0₋(∞) C(max), T(max), t(½) CL/F and apparent volume of distribution during the terminal phase Xz; urinary parameters were the amount of aclidinium or acid or alcohol metabolite excreted in urine, the percentage of the dose excreted in urine (fe), and renal clearance (CL(R)). Tolerability was assessed using physical examination, vital signs, 12-lead ECG recordings, laboratory tests, and adverse-event (AE) reports. The Wilcoxon rank sum test was used to compare the median PK values between the normal and impaired renal function groups. Pearson correlation coefficients and linear regression models were used to analyze the relationship between creatinine clearance and AUC0₋(∞) and between creatinine clearance and CL(R) for aclidinium and its metabolites. RESULTS: A total of 16 men and 8 women were included in the study. All participants were white; mean (SD) age was 55 (10.7) years and weight was 70.8 (9.2) kg. Aclidinium Cmax was observed in plasma by 5 minutes after dosing (ie, median Tmax) and did not differ significantly among the renal function groups. Plasma concentrations of aclidinium declined after reaching Cmax, with median t(½) values ranging from 2.07 to 4.18 hours across all renal function groups. Most of the individual t(½) values were between 1.5 and 3.5 hours, regardless of the degree of renal insufficiency. No significant relationship between AUC0₋(∞)) and creatinine clearance was observed (Pearson correlation coefficient = -0.0446; P = NS). Urinary excretion of aclidinium was very low, with a mean 0.090% (median 0.078%) of the dose recovered from the urine in participants with normal renal function. Eight AEs were reported in 7 participants after drug administration; all were mild to moderate in severity and resolved spontaneously. There were no serious drug-related AEs and no deaths. CONCLUSIONS: The plasma PK parameters of aclidinium bromide were not significantly altered after a single inhaled dose of aclidinium bromide 400 µg in this small group of patients with various degrees of impaired renal function. The very low urinary excretion of aclidinium in all renal function groups indicates that renal function plays a minor role in aclidinium plasma clearance. Aclidinium appeared well tolerated in the population studied. These results suggest that aclidinium dose adjustment on the basis of renal function may not be necessary.

[Retrospective study of two cases of acute datura poisoning].

We saw 2 cases of datura poisoning, with the main complaint of impaired consciousness, which were brought to the emergency department of this hospital. The poisonous constituent of the datura is tropane alkaloid, which is said to have a short half-life period that makes it difficult to determine its quantity in the blood or urine. We stored the urine and blood specimens taken when the patients were brought in and sent them for analysis at a public health center a few days later. The determined amounts for both patients are as follows. Case 1: serum atropine, 70 ng/mL; scopolamine, 210 ng/mL; urine atropine, 0.34 mg/L; scopolamine, 0.11 mg/L. Case 2 : serum atropine, 60 ng/ mL ; scopolamine, 380 ng/mL ; urine atropine, 0.22 mg/L ; scopolamine, 0.14 mg/L. We attempted to compare these results with literature on the possible measurement of a determined amount in light of comparative considerations of the correlation between blood concentration and clinical conditions, but encountered some difficulties due to the lack of available reports. However, as our own experiment cases are retrospective we report them as valuable cases in which it is possible to obtain a determined amount of tropane alkaloid.

Quantitative analysis of tropane alkaloids in biological materials by gas chromatography-mass spectrometry.

A simple and rapid method for quantitation of tropane alkaloids in biological materials has been developed using an Extrelut column with gas chromatography-mass spectrometry (GC-MS). Biological materials (serum and urine) were mixed with a borate buffer and then applied to an Extrelut column. The adsorbed tropane alkaloids were eluted with dichloromethane before a GC-MS analysis. Atropine-d(3) was used as an internal standard. The extracted tropane alkaloids were converted to trimethylsilyl derivatives prior to GC analysis, to improve the instability of tropane alkaloids from heating and the property of them for a GC column. The recoveries of the compounds, which had been spiked to biological materials, were more than 80%. The GC separation of the derivatives from endogenous impurities was generally satisfactory with the use of a semi-polar capillary column. Tropane alkaloids showed excellent linearity in the range of 10-5000 ng/ml and the limit of detection was 5.0 ng/ml for biological materials. The present method is simple and more rapid than those previously reported, and was applied to a poisoning case. It is useful for the routine analysis of tropane alkaloids in cases of suspected tropane alkaloids poisoning.

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%.

Human biodistribution and dosimetry of [123I]FP-CIT: a potent radioligand for imaging of dopamine transporters.

This study reports on the biodistribution and radiation dosimetry of iodine-123-labelled N-omega-(flu- oropropyl)-2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([123I]FP-CIT), a promising radioligand for the imaging of dopamine transporters. In 12 healthy volunteers, conjugate whole-body scans were performed up to 48 h following intravenous injection of approximately 100 MBq [123I]FP-CIT. Attenuation correction was performed using a transmission whole-body scan obtained prior to injection of the radioligand, employing a 123I flood source. Blood samples were taken and urine was freely collected up to 48 h after injection of the radiotracer. For each subject, the percentage of injected activity measured in regions of interest over brain, striatum, lungs and liver were fitted to a multicompartmental model to give time-activity curves. The cumulative urine activity curve was used to model the urinary excretion rate and, indirectly, to predict faecal excretion. Using the MIRD method, nine source organs were considered in estimating absorbed radiation doses for organs of the body. The images showed rapid lung uptake and hepatobiliary excretion. Diffuse uptake and retention of activity was seen in the brain, especially in the striatum. At 48 h following the injection of [123I]FP-CIT, mean measured urine excretion was 60%+/-9% (SD), and mean predicted excretion in faeces was 14%+/-1%. In general, the striatum received the highest absorbed dose (average 0.23 mGy/MBq), followed by the urinary bladder wall (average 0.054 mGy/MBq) and lungs (average 0.043 mGy/MBq). The average effective dose equivalent of [123I]FP-CIT was estimated to be 0.024 mSv/MBq. The amount of [123I]FP-CIT required for adequate dopamine transporter imaging results in an acceptable effective dose equivalent to the patient.

Phase I and II metabolites of benztropine in rat urine and bile.

Following oral administration of benztropine (IO mg/kg, body weight), the phase I metabolites, benztropine N-oxide, N-desmethylbenztropine, tropine, 4'-hydroxybenz- tropine, N-desmethyl-4'-hydroxybenztropine, 4'-hydroxvbenztropine N-oxide and methoxy-4'-hydroxybenztropine, together with unmetabolized benztropine, were isolated and identified in rat urine and bile by GC-electron impact mass spectrometry (EI GC/MS), microcolumn LC-electrospray mass spectrometry (ES LC/MS) and hplc followed by MS analysis. The mass spectra and chromatographic properties of isolated N-desmethylbenztropine, benztropine N-oxide and tropine were confirmed by comparison with authentic reference standards. Sufficient quantities of 4'-hydroxybenztropine and N-desmethyl-4'-hydroxybenztropine were isolated from the urine by tlc and examined by 1H-nmr, ES/MS and EI/MS. The structure of the methoxy-4'-hydroxybenztropine metabolite was determined by EI/MS. 4'-Hydroxybenztropine N-oxide was identified by reacting it with a reducing agent, titanous chloride, to form 4'-hydroxybenztropine, which was then confirmed by comparing its EI/MS and ES/MS behaviour with a previously isolated and 1H-nmr-authenticated sample. In addition, four intact glucuronide conjugates of benztropine were also characterized in bile and urine as phase II metabolites, including 4'-O-glucuronylbenzotropine, N-desmethyl-4'-O-glucuronylbenztropine, methoxy-4'-O-glucuronylbenztropine and 4'- O-glucuronylbenztropine N-oxide by hplc followed by ES/MS analysis. These results provide the first direct evidence of the presence of these metabolites of benztropine in rat.

Identification of urinary metabolites of 8-methyl-8-azabicyclo-[3,2,1] octan-3-yl 3,5-dichlorobenzoate (MDL 72,222) in the dog and monkey.

The metabolism of 8-methyl-8-azabicyclo- 3,2,1]octan-3-yl 3,5-dichlorobenzoate (MDL 72,222) was studied in the dog and monkey. Four urinary metabolites were detected by HPLC, HPLC/MS, and GC/MS, and were identified by comparison to authentic standards. The major metabolite in the dog, approximately 41% of the administered dose excreted between 0 and 120 hr, was the MDL 72,222-N-oxide. On the other hand, the major metabolite in the monkey was the glycine conjugate of 3,5-dichlorobenzoic acid (greater than 56% of the dose). Seven percent of the dose in the monkey urine was free 3,5-dichlorobenzoic acid. N-Desmethyl MDL 72,222 was present at 2.5 and 1% in the dog and monkey, respectively. Very little (less than 1%) of the parent compound was found in urine. The major pathways of metabolism of MDL 72,222 are N-oxidation, N-demethylation, ester hydrolysis, and amino acid conjugation.

Determination of the quaternary compound ciclotropium in human biological material after hydrolysis and derivatization with the fluorophor flunoxaprofen chloride.

The quantitative determination of the quaternary spasmolytic compound ciclotropium and its metabolite N-isopropyltropinium is described for human plasma and urine. The analytical procedure consists of ion-pair extraction from biological material, alkaline hydrolysis, subsequent derivatization with the fluorophor flunoxaprofen chloride and separation by high-performance liquid chromatography on a reversed-phase column with fluorimetric monitoring. The detection limits of 0.5 ng/ml in plasma and 10 ng/ml in urine at signal-to-noise ratios higher than 3 permit the determination of pharmacokinetic parameters after therapeutic doses.

[An evaluation of methods for isolating antiparkinson preparations from cadaveric material]. / Otsenka metodov vydeleniia nekotorykh protivoparkinsonicheskikh preparatov iz trupnogo materiala.

Methods of amedine, amyzyl, and tropacine isolation from cadaveric material, blood and urine were developed. They ensure higher output of the given substances as compared with conventional methods of isolation of toxicologically significant substances. An extraction-photometric method was developed for estimation of amedine, amyzyl, and tropacine in the cadaveric material.

The metabolism of atropine in man.

A metabolic pattern of atropine in man, based on the detection of radiolabelled products in urine by high performance liquid chromatography after administration of [3H]atropine sulphate to a normal volunteer is proposed. Noratropine (24%), atropine-N-oxide (equatorial isomer) (15%), tropine (2%) and tropic acid (3%) appear to be the major metabolites, while 50% of the administered dose is excreted as apparently unchanged atropine. No conjugates were detectable. Evidence that atropine is present as (+)-hyoscyamine was found, suggesting that stereoselective metabolism of atropine probably occurs.