Synthesis of magnetic halloysite nanotube-based molecularly imprinted polymers for sensitive spectrophotometric detection of metoclopramide in urine samples.

A novel molecularly imprinted polymer was synthesized on magnetic halloysite nanotube via surface initiated reversible addition-fragmentation chain transfer polymerization in the presence of 2-aminoethylmethacrylamide, 2-Cyano-2-propyl benzodithioate, ethylene glycol dimethacrylate (EGDMA) and azobis(isobutyronitrile) for sensitive and selective spectrophotometric determination of metoclopramide in urine samples. The synthesized imprinted polymer was characterized by several surface characterization techniques and the results indicated there was a thin polymer network on the magnetic halloysite nanotube. The rebinding properties of the molecularly imprinted magnetic halloysite nanotube were also investigated in detail and the maximum adsorption capacity and imprinting factor were found to be 37.8 mg/g and 4.51, respectively. The application of the proposed method was carried out by enrichment and spectrophotometric determination of metoclopramide via formation of a charge transfer complex between picric acid and eluted metoclopramide. Under the optimized conditions, the calibration curve was linear in the concentration range of 5.0-150.0 ng/mL and the limit of detection and the limit of quantification were calculated to be 1.5 ng/mL and 4.95 ng/mL, respectively. The inter-day and intra-day precisions were below 5% and recoveries were between 92.8% and 99.2%. The results showed that the proposed method increased the sensitivity and selectivity for spectrophotometric determination of metoclopramide.

An accelerated background subtraction algorithm for processing high-resolution MS data and its application to metabolite identification.

BACKGROUND: Metabolite identification without radiolabeled compound is often challenging because of interference of matrix-related components. RESULTS: A novel and an effective background subtraction algorithm (A-BgS) has been developed to process high-resolution mass spectral data that can selectively remove matrix-related components. The use of a graphics processing unit with a multicore central processing unit enhanced processing speed several 1000-fold compared with a single central processing unit. A-BgS algorithm effectively removes background peaks from the mass spectra of biological matrices as demonstrated by the identification of metabolites of delavirdine and metoclopramide. CONCLUSION: The A-BgS algorithm is fast, user friendly and provides reliable removal of matrix-related ions from biological samples, and thus can be very helpful in detection and identification of in vivo and in vitro metabolites.

Sensitive flow-injection spectrophotometric analysis of bromopride.

A flow injection spectrophotometric procedure employing merging zones is proposed for direct bromopride determination in pharmaceutical formulations and biological fluids. The proposed method is based on the reaction between bromopride and p-dimethylaminocinnamaldehyde (p-DAC) in acid medium, in the presence of sodium dodecyl sulfate (SDS), resulting in formation of a violet product (λmax=565nm). Experimental design methodologies were used to optimize the experimental conditions. The Beer-Lambert law was obeyed in a bromopride concentration range of 3.63×10(-7) to 2.90×10(-5)molL(-1), with a correlation coefficient (r) of 0.9999. The limits of detection and quantification were 1.07×10(-7) and 3.57×10(-7)molL(-1), respectively. The proposed method was successfully applied to the determination of bromopride in pharmaceuticals and human urine, and recoveries of the drug from these media were in the ranges 99.6-101.2% and 98.6-102.1%, respectively. This new flow injection procedure does not require any sample pretreatment steps.

Effect of metoclopramide on the excretion rate of paracetamol using HPLC-DAD.

A simple, precise, accurate and robust high-performance liquid chromatography assay was developed and validated for the simultaneous analysis of metoclopramide and paracetamol in human urine. The drugs were isolated from urine samples by solid-phase extraction using C8 cartridges, then analyzed on a C18 reversed-phase column using a mixture of aqueous phase (water containing 0.2% TEA adjusted to pH 3 using ortho-phosphoric acid) and methanol in a ratio of 80:20 (v/v). The method was found to be linear for both drugs in a concentration range of 0.5 to 160 µg/mL using a concentration of 10 µg/mL of internal standard (theophylline) in urine samples (r > 0.999). The accuracy of the method was higher than 91.73% (percentage of the grand mean of recoveries) and the precision was lower than 3.4% (overall percentage of relative standard deviation) for both metoclopramide and paracetamol. The method was applied to the determination of the drugs in urine samples obtained from male volunteers, following the administration of two formulations, one containing paracetamol alone (Paracetamol(®)) and the other containing a mixture of paracetamol and metoclopramide (Migracicid(®)). Determination of the drugs was conducted and the effect of increasing the rate of absorption, consequently increasing the mean urinary excretion of paracetamol due to the presence of metoclopramide in the pharmaceutical formulation, was recorded.

Identification of novel metoclopramide metabolites in humans: in vitro and in vivo studies.

Metoclopramide (MCP) is frequently used to treat gastroparesis. Previous studies have documented MCP metabolism, but systematic structural identification of metabolites has not been performed. The aim of this study was to better understand MCP metabolism in humans. For examination of in vivo metabolism, a single oral 20-mg MCP dose was administered to eight healthy male volunteers, followed by complete urine collection over 24 h. In vitro incubations were performed in human liver microsomes (HLM) to characterize metabolism via cytochromes P450 and UDP-glucuronosyltransferases and in human liver cytosol for metabolism via sulfotransferases. Urine and subcellular incubations were analyzed for MCP metabolites on a mass spectrometer with accurate mass measurement capability. Five MCP metabolites were detected in vivo, and five additional metabolites were detected in vitro. The five metabolites of MCP identified both in vitro and in vivo were an N-O-glucuronide (M1), an N-sulfate (M2), a des-ethyl metabolite (M3), a hydroxylated metabolite (M4), and an oxidative deaminated metabolite (M5). To our knowledge, metabolites M1 and M4 have not been reported previously. M2 urinary levels varied 22-fold and M3 levels varied 16-fold among eight subjects. In vitro studies in HLM revealed the following additional metabolites: two ether glucuronides (M6 and M8), possibly on the phenyl ring after oxidation, an N-glucuronide (M7), a carbamic acid (M9), and a nitro metabolite (M10). Metabolites M6 to M10 have not been reported previously. In conclusion, this study describes the identification of MCP metabolites in vivo and in vitro in humans.

Novel molecularly imprinted polymers for the selective extraction and determination of metoclopramide in human serum and urine samples using high-performance liquid chromatography.

This work was performed in order to study the possibilities in using molecularly imprinted polymers (MIPs) as sorbent material in solid-phase extraction (MISPE) for the sample clean-up technique for the determination of metoclopramide (MCP) in biological fluids. The effective factors influencing the bulk polymerization have been studied. Molecular recognition properties, binding capability and selectivity of the molecularly imprinted polymers (MIPs) were evaluated and the results revealed the obtained MIPs have high affinity for MCP in aqueous environment. The optimal conditions for solid-phase extraction (SPE) consisted of conditioning with 1 mL of methanol and 1 mL of deionized water at neutral pH, loading with 1 mL of the water sample (50 microg L(-1)) at pH 8.5, washing using 1 mL of acetone and elution with 2x 1 mL methanol/acetic acid (10/1, v/v). After optimization of SPE procedure, the MIP was then directly used to selectively extract the target drug from human serum and urine with an extraction recovery of more than 90%. Chromatograms of the eluate solutions show an efficient clean-up, which supports the potential of MISPE for clean-up of trace amounts of MCP from serum and urine. The limits of detection of MCP in human serum and urine were 3 and 1.2 microg L(-1), respectively.

Electrogenerated chemiluminescence sensor for metoclopramide determination based on Ru(bpy)3 2+-doped silica nanoparticles dispersed in Nafion on glassy carbon electrode.

A novel method for the determination of metoclopramide (MCP) using electrogenerated chemiluminescence (ECL) is presented. A tris(2,2'-bipyridyl)dichlororuthenium(II) (Ru(bpy)3(2+))-doped silica (RuDS) nanoparticle/perfluoinated ion-exchange resin (Nafion) with nanocomposite membrane modified glassy carbon electrode (GCE) is used. The Ru(bpy)3(2+) encapsulation interior of the silica nanoparticle maintains its electrochemical activities and also reduces Ru(bpy)3(2+) leaching from the silica matrix when immersed in water due to the electrostatic interaction. The analytical performance of this ECL sensor for MCP is shown in detail. Under optimal experimental conditions, it has good linearity in the concentration range from 2 x 10(-8)mol/L to 1 x 10(-5)mol/L (R=0.9989) with a detection limit of 7 x 10(-9)mol/L. The relative standard deviation (n=11) is 3.2% for detecting 1.2 x 10(-6)mol/L MCP. The recoveries are in the range of 97.0-104.4% for sample measurements by standard-addition method. This method has been applied successfully to determine MCP in pharmaceutical preparations and in human urine. Statistical analysis (Student's t-test and variance ratio F-test) of the obtained results show no significant difference between this proposed method and the reference method.

Square wave anodic stripping voltammetric determination of metoclopramide in tablet and urine at carbon paste electrode.

A simple, reliable and selective square wave anodic stripping (SWAS) voltammetric method at carbon paste electrode (CPE) of metoclopramide hydrochloride (MCP) in pharmaceutical dosage forms (tablet) and in biological fluids (spiked and real urine samples) has been developed and evaluated. Different parameters such as medium, supporting electrolyte, pH, accumulation potential, scan rate, accumulation time and ionic strength, were tested to optimize the conditions for the determination of MCP. The adsorbed form is oxidized irreversibly under optimal conditions, viz., 0.4M HCl-sodium acetate buffer (pH approximately 6.2), 0.2M KCl, a linear concentration ranges from 0.067 to 0.336, 0.067 to 0.269 and 0.067 to 0.269 ng/mL of MCP, at accumulation times 60, 120 and 180 s, respectively, can be determined successfully. The interferences of some common excipients and some metal ions were studied. The standard addition method was used to determine the MCP in pure solutions, tablets and in biological fluids with satisfactory results. The data obtained are compared with the standard official method.

Synthesis of a metabolite of metoclopramide and its detection in human urine.

The synthesis of 2-(4-amino-5-chloro-2-methoxybenzamido)acetic acid 2, a metabolite of metoclopramide 1, has been accomplished through the coupling of 4-amino-5-chloro-2-methoxybenzoic acid 4 with glycine benzyl ester followed by a catalytic hydrogenation. Such a metabolite could not be detected directly in the human urines but only after its transformation into the corresponding methyl ester 6. Compound 6 was prepared both by condensing acid 4 with glycine methyl ester and by reacting acid 2 with diazomethane. HPLC analyses of biological samples were therefore performed after treatment with diazomethane. In five healthy volunteers, the percentage of 6 spanned the range 0.6-1.2%.

Identification of metoclopramide metabolites in the urine of cattle by gas chromatography-mass spectrometry and high-performance liquid chromatography-photodiode array detection.

The urinary metabolites of metoclopramide (4-amino-5-chloro-N-[2-diethylaminoethyl]-2-methoxybenzamide) were identified in cows. The drug was administered intravenously, voided urine was collected, and individual urine extracts were analysed by gas chromatography-mass spectrometry and high-performance liquid chromatography-photodiode array detection. The parent compound and one major metabolite (4-amino-5-chloro-N-[2-(ethylamino)ethyl]-2-methoxybenzamide) were common to all individuals. In addition to the parent and major metabolite, a second, minor metabolite was identified in two cows as 4-amino-5-chloro-N-[2-(diethylamino)ethyl]-2-hydroxybenzamide. The identity of the minor metabolite was confirmed by comparison with a standard synthesized by a new method. Metabolite identification and characterization in food animal species allows the design of safety and environmental impact studies and relative metabolite ratios between dose treatment groups.

Determination of metoclopramide in the serum and urine of cattle.

Metoclopramide, a dopamine-2 agent, has been shown to be useful in the antagonism of fescue toxicosis in grazing steers. The determination of this drug is described for the purpose of pharmacokinetic study and consideration of potential delivery devices to combat this economically significant condition.

Quantitation of a novel antiemetic (ADR-851) in plasma and urine by reversed-phase high-performance liquid chromatography with fluorescence detection.

A sensitive and specific bioanalytical method for quantitation of a novel antiemetic (ADR-851) in plasma and urine has been developed and validated. The drug and internal standard (metoclopramide) are extracted from the plasma matrix by solid-phase extraction on cyanopropyl bonded-phase columns. After extraction, samples are separated by isocratic reversed-phase high-performance liquid chromatography. The parent drug, internal standard and a yet unidentified metabolite are detected by fluorescence. The method requires 1.0 ml of plasma or 0.1 ml of urine and has a lower limit of quantitation of 2 ng/ml with 10.9% relative standard deviation (R.S.D.). Method linearity has been established over a 2-800 ng/ml range when 1.0 ml of plasma is used. The intra- and inter-day imprecisions for the method are typically better than 6% and 11% R.S.D., respectively, in both plasma and urine over the entire dynamic range. The pooled estimate of bias is less than 5% and attests to the excellent accuracy.

Column-switching liquid chromatographic determination of ML-1035 sulphoxide and its sulphone and sulphide metabolites in rat urine.

A fully automated column-switching LC assay has been developed for the simultaneous determination of a gastroprokinetic agent, ML-1035 sulphoxide, and its sulphone and sulphide metabolites in rat urine. ML-1035 Sulphoxide is a metoclopramide analogue. The method involved direct injection of a diluted urine sample into a CN extraction column for sample clean-up. Polar urine components, including proteins, were flushed to waste. The retained compounds were then eluted onto a C8 analytical column for further separation and analysis by fluorescence detection. After the subsequent washing and re-equilibration with a sequence of three solvent mixtures, the extraction column was ready for the next injection. The recovery of the compounds from the extraction column was 85-90%. The limit of quantitation for all compounds of interest was 25 ng ml-1 or lower, using a 100 microliters specimen of urine. Good inter-day precision (2.1-10.0%), accuracy (0.3-18.0%), and linearity were obtained for all compounds over a range of 25-1000 ng ml-1. The applicability of the LC method was validated with urine samples from rats that had received ML-1035 sulphoxide.

Liquid chromatographic analysis of alizapride and metoclopramide in human plasma and urine using solid-phase extraction.

A universal rapid, sensitive and selective high-performance liquid chromatographic method with UV detection at 230 nm has been developed for the determination of benzamide drugs in human plasma and urine. Sample pretreatment is carried out using solid-phase extraction columns, resulting in very high extraction recoveries of the compounds investigated (alizapride, metoclopramide, alpiropride, amisulpride). The detector response is linear from 25 to 10,000 ng/ml, and the detection limit is 3 ng/ml for alizapride and 10 ng/ml for metoclopramide. The proposed method is highly suitable for pharmacokinetic studies and for drug monitoring.

Determination of metoclopramide and its glucuronide and sulphate conjugates in human biological fluids (plasma, urine and bile) by ion-pair high-performance liquid chromatography.

Metoclopramide was determined in human biological fluids (plasma, urine and bile) by reversed-phase ion-pair high-performance liquid chromatography using a newly introduced cyanopropyl column. The method is precise, selective and sensitive: the mean recoveries of metoclopramide from plasma, urine and bile were 74.4, 99.1 and 85.9%, respectively; the mean within- and between-run coefficients of variation were, respectively, 0.8 and 8.5% for plasma and 2.0 and 8.2% for urine at the drug concentration of 100 ng/ml, and 2.3 and 11.2% for bile at the concentration of 20 ng/ml; the lower detection limit was 2 ng/ml for 1 ml of each biological fluid. Enzymic hydrolysis of a urine or bile specimen was used in the identification of metoclopramide, as well as its glucuronide and sulphate conjugates, from the human samples. A preliminary study on metoclopramide determinations from plasma and urine samples of a healthy subject and from bile samples of a patient demonstrated the clinical applicability of the method for therapeutic monitoring and pharmacokinetic studies.

Pharmacokinetics and bioavailability of the anti-emetic agent bromopride.

The pharmacokinetics of bromopride, an anti-emetic agent chemically related to metoclopramide, has been investigated in normal human subjects. After intravenous bolus doses of 10 mg, a one-compartment open model appeared adequate to describe the plasma drug concentration data. The systemic clearance of bromopride was 899 ml min-1 +/- 22 per cent CV, the volume of distribution was 2151 +/- 16 per cent CV, and the elimination half-life was 2.9 h +/- 21 per cent CV. Over a wide drug concentration range of up to 650 ng ml-1, bromopride was only 40 per cent bound to plasma proteins. The systemic availability of orally and intramuscularly administered solution doses of 20 mg of bromopride was 54 per cent and 78 per cent, respectively. Formulation of bromopride as the solid material in capsules delayed absorption but did not affect the extent of drug bioavailability. The pharmacokinetics of bromopride appeared similar to that of metoclopramide. No evidence for non-linear kinetics was found when bromopride was administered orally in the dose range 10-30 mg: after single oral doses of 10, 20, and 30 mg, peak mean plasma drug concentrations were 20 ng ml-1 +/- 32 per cent CV, 38 ng ml-1 +/- 16 per cent CV, and 64 ng ml-1 +/- 23 per cent CV, respectively.

Metoclopramide kinetics in patients with impaired renal function and clearance by hemodialysis.

Metoclopramide kinetics were examined in 24 adult patients with diminished renal function and in eight healthy subjects with normal renal function. Creatinine clearance correlated with metoclopramide plasma clearance, renal clearance, nonrenal clearance, and elimination t1/2. Regardless of renal function, renal clearance accounted for less than or equal to 21% of total plasma clearance. Nonrenal clearance was reduced in patients and accounted for most of the reduction in plasma clearance. The comparatively small plasma clearances in patients imply that maintenance doses should be reduced accordingly to avoid drug cumulation. Metoclopramide clearance by hemodialysis was also assessed in four patients. Metoclopramide losses from hemodialysis were relatively small compared to estimates of total body metoclopramide stores. Compensatory dosage increases are probably unnecessary for most patients. These data also suggest that hemodialysis is not likely to be effective in metoclopramide overdose.

Determination of bromopride in human plasma and urine by high-performance liquid chromatography.

Bromopride was measured in plasma and urine using reversed-phase high-performance liquid chromatography employing ultraviolet absorption detection. The limit of detection in plasma was 2 ng/ml, sufficient for pharmacokinetic studies of the drug. Plasma concentrations of bromopride reached mean peak levels (55 ng/ml) at 1 h after single oral doses of 20 mg and declined with a half-life of 4.9 h. Less than a mean of 10% of an oral dose was excreted unchanged in the urine. The assay could also be used to measure metoclopramide in these bio-fluids.