On-chip ion pair-based dispersive liquid-liquid extraction for quantitative determination of histamine H2 receptor antagonist drugs in human urine.

In the present work, an ion-pair based dispersive liquid-liquid microextraction was performed on a centrifugal chip for the first time. The entire DLLME procedure, including flow direction, desperation, and sedimentation of the extracting phase, can be fulfilled automatically on a solitary chip. The chip was made of Poly(methyl methacrylate) (PMMA) and was of two units for two parallel extractions, each consisting of three chambers (for the sample solution, extracting solvents, and sedimentation). As the chip rotated, fluids flowed within the chip, and the dispersion, mixing, extraction, and sedimentation of the final phase were performed on the chip by simply adjusting the spin speed. Determination of two histamine H2 receptor antagonist drugs, cimetidine and ranitidine, as the model analytes from the urine samples was done using the developed on-chip ion-pair based DLLME method followed by an HPLC-UV. The effective parameters on the extraction efficiency of the model analytes were investigated and optimized using the one variable at a time method. Under optimized conditions, the calibration curve was linear in the range of 15-2000 µg L-1 with a coefficient of determination (R2) more than 0.9987. The relative standard deviations (RSD %) for extraction and determination of the analytes were less than 3.7% based on five replicated measurements. LODs less than 10.0 µg L-1 and preconcentration factors higher than 39-fold were obtained for both of the model analytes. The proposed chip enjoys the advantages of both the DLLME method and miniaturization on a centrifugal chip.

Development and validation of a sensitive LC-MS/MS assay for the quantification of nizatidine in human plasma and urine and its application to pharmacokinetic study.

We developed and validated a high performance liquid chromatographic method coupled with triple quadrupole mass spectrometry for analysis of nizatidine in human plasma and urine. The biological samples were precipitated with methanol before separation on an Agilent Eclipse Plus C18 column (100mm×46mm, 5µm) with a mixture of methanol and water (95:5, plus 5mM ammonium formate) as the mobile phase at 0.5mL/min. Detection was performed using multiple reaction monitoring modes via electrospray ionization (ESI) at m/z 332.1→155.1 (for nizatidine) and m/z 335.1→155.1 (for [(2)H3]-nizatidine, the internal standard). The linear response range was 5-2000ng/mL and 0.5-80µg/mL for human plasma and urine, with the lower limits of quantification of 5ng/mL and 0.5µg/mL, respectively. The method was validated according to the biological method validation guidelines of the Food and Drug Administration and proved acceptable. This newly developed analytical method was successfully applied in a pharmacokinetic study following single oral administration of a 150mg nizatidine capsule in to 16 healthy Chinese subjects. Maximum and endpoint concentrations in plasma and urine were quantifiable, suggesting our method is appropriate for routine pharmacokinetic analysis.

Ranitidine interference with standard amphetamine immunoassay.

BACKGROUND: We recently encountered several cases of possible false positive results of amphetamine on the Beckman Coulter AMPH assay, but not on the Siemens EMIT II Plus assay. Our clinical chart review suggested that ranitidine interference may be responsible for the false positive results of the AMPH assays. METHODS: Blank urine specimens spiked with ranitidine concentrations ranging from 5µg/ml to 5mg/ml were analyzed on both the AMPH and EMIT II Plus assays. To examine if the false positive results were due to assay specific reagent/sample ratios, we prepared 3 different sample to reagent ratios and analyzed them for amphetamine reaction rates on both assays. RESULTS: Ranitidine at 160µg/ml caused a positive interference on the AMPH assay. No interference was observed by ranitidine on the EMIT II Plus assay. Specifically, the sample to reagent ratios tested neither eliminated the positive inference on the AMP assay nor introduced an interference on the EMIT II Plus assay. CONCLUSIONS: Unlike the EMIT II Plus assay, the AMPH assay has cross-activity with ranitidine, which is independent of sample to reagent ratio.

Spectrofluorimetric analysis of famotidine in pharmaceutical preparations and biological fluids by derivatization with benzoin.

A sensitive and simple spectrofluorimetric method has been developed for the analysis of famotidine, from pharmaceutical preparations and biological fluids after derivatization with benzoin. The reaction was carried out in alkaline medium with measurement of fluorescence intensity at 446 nm with excitation wavelength at 286 nm. Linear calibration was obtained with 0.5-15 µg/ml with coefficient of determination (r(2)) 0.997. The factors affecting the fluorescence intensity were optimized. The pharmaceutical additives and amino acid did not interfere in the determination. The mean percentage recovery (n=4) calculated by standard addition from pharmaceutical preparation was 94.8-98.2% with relative standard deviation (RSD) 1.56-3.34% and recovery from deproteinized spiked serum and urine of healthy volunteers was 98.6-98.9% and 98.0-98.4% with RSD 0.34-0.84% and 0.29-0.87% respectively.

Determination of ranitidine, nizatidine, and cimetidine by a sensitive fluorescent probe.

A validated, simple, and sensitive fluorescence quenching method for the determination of ranitidine, nizatidine, and cimetidine in tablets and biological fluids is presented. This is the first single fluorescence method reported for the analysis of all three H(2) antagonists. The competitive reaction between the investigated drug and the palmatine probe for the occupancy of the cucurbit[7]uril (CB[7]) cavity was studied using spectrofluorometry. CB[7] was found to react with the probe to form a stable complex. The fluorescence intensity of the complex was also enhanced greatly. However, the addition of the drug dramatically quenched the fluorescence intensity of the complex. Accordingly, a new fluorescence quenching method for the determination of the studied drugs was established. The different experimental parameters affecting the fluorescence quenching intensity were studied carefully. At optimum reaction conditions, the rectilinear calibration graphs between the fluorescence quenching values (ΔF) and the medicament concentration were obtained in the concentration range of 0.04-1.9 µg mL(-1) for the investigated drugs. The limits of detection ranged from 0.013 to 0.030 µg mL(-1) at 495 nm using an excitation wavelength of 343 nm. The proposed method can be used for the determination of the three H(2) antagonists in raw materials, dosage forms and biological fluids.

Polyethylene glycol 400 enhances the bioavailability of a BCS class III drug (ranitidine) in male subjects but not females.

PURPOSE: The aim of this study was to investigate the effects of different doses of polyethylene glycol 400 (PEG 400) on the bioavailability of ranitidine in male and female subjects. METHOD: Ranitidine (150 mg) was dissolved in 150 ml water with 0 (control), 0.5, 0.75, 1, 1.25 or 1.5 g PEG 400 and administered to 12 healthy human volunteers (six males and six females) in a randomized order. The cumulative amount of ranitidine and its metabolites excreted in urine over 24 h was determined for each treatment using a validated HPLC method. RESULTS: In the male volunteers, the mean cumulative amount of ranitidine excreted in the presence of 0, 0.5, 0.75, 1, 1.25 and 1.5 g PEG 400 were 35, 47, 57, 52, 50 and 37 mg respectively. These correspond to increases in bioavailability of 34%, 63%, 49%, 43% and 6% over the control treatment. In the female subjects, the mean cumulative quantity of ranitidine excretion in the absence and presence of increasing amounts of PEG 400 were 38, 29, 35, 33, 33 and 33 mg, corresponding to decreases in bioavailability of 24%, 8%, 13%, 13% and 13% compared to the control. The metabolite excretion profiles followed a similar trend to the parent drug at all concentrations of PEG 400. CONCLUSIONS: All doses of PEG 400 enhanced the bioavailability of ranitidine in male subjects but not females, with the most pronounced effect in males noted with the 0.75 g dose of PEG 400 (63% increase in bioavailability compared to control, p < 0.05). These findings have significant implications for the use of PEG 400 in drug development and also highlight the importance of gender studies in pharmacokinetics.

Simple and universal HPLC-UV method to determine cimetidine, ranitidine, famotidine and nizatidine in urine: application to the analysis of ranitidine and its metabolites in human volunteers.

A validated, simple and universal HPLC-UV method for the determination of cimetidine, famotidine, nizatidine and ranitidine in human urine is presented. This is the first single HPLC method reported for the analysis of all four H(2) antagonists in human biological samples. This method was also utilized for the analysis of ranitidine and its metabolites in human urine. All calibration curves showed good linear regression (r(2)>0.9960) within test ranges. The method showed good precision and accuracy with overall intra- and inter-day variations of 0.2-13.6% and 0.2-12.1%, respectively. Separation of ranitidine and its metabolites using this assay provided significantly improved resolution, precision and accuracy compared to previously reported methods. The assay was successfully applied to a human volunteer study using ranitidine as the model compound.

Determination of ranitidine in urine by capillary electrophoresis-electrochemiluminescent detection.

The fast analysis of ranitidine is of clinical importance in understanding its efficiency and a patient's treatment history. In this paper, a novel determination method for ranitidine based on capillary electrophoresis-electrochemiluminescence detection is described. The conditions affecting separation and detection were investigated in detail. End-column detection of ranitidine in 5 mM Ru(bpy)(3)(2+) solution at applied voltage of 1.20 V was performed. Favorable ECL intensity with higher column efficiency was achieved by electrokinetic injection for 10s at 10 kV. The R.S.D. values of ECL intensity and migration time were 6.38 and 1.84% for 10(-4) M and 6.01 and 0.60% for 10(-5) M, respectively. A detection limit of 7 x 10(-8) M (S/N=3) was achieved. The proposed method was applied satisfactorily to the determination of ranitidine in urine in 6 min.

Determination of ebrotidine and its metabolites by micellar electrokinetic capillary chromatography.

Ebrotidine and its potential metabolites were determined by micellar electrokinetic capillary chromatogaphy (MECC) using sodium dodecylsulfate (SDS) as surfactant. The influences of buffer composition, SDS concentration and addition of a neutral surfactant such as Brij 35 were studied. A 40 mM phosphate buffer at pH 7.50 containing 50 mM of SDS was selected as carrier electrolyte, and provided the optimum separation with regard to resolution and migration time. Linear calibration curves over the range studied (5.0-50 microg ml(-1)), limits of detection between 0.25 and 2.0 microg ml(-1) and run-to-run precision lower than 10% were obtained. The MECC method was applied to the determinaton of these compounds in spiked human urine.

Development of a sensitive high-performance thin-layer chromatography method for estimation of ranitidine in urine and its application for bioequivalence decision for ranitidine tablet formulations.

A sensitive and simple HPTLC method was developed for estimation of ranitidine in human urine. The drug was extracted from urine after basification using dichloromethane. Dichloromethane extract was spotted on silica gel 60 F254 TLC plate and was developed in a mixture of ethyl acetate-methanol-ammonia (35:10:5 v/v) as the mobile phase and scanned at 320 nm. The RF value obtained for the drug was 0.67 +/- 0.03. The method was validated in terms of linearity (50-400 ng/spot), precision and accuracy. The average recovery of ranitidine from urine was 89.35%. The proposed method was applied to evaluate bioequivalence of two marketed ranitidine tablet formulations (150 mg, Formulation I and Formulation 2) using a crossover design by comparing urinary excretion data for unchanged ranitidine in six healthy volunteers. Various pharmacokinetic parameters like peak excretion rate [(dAU/dt)max], time for peak excretion rate (tmax), AUC0-24, AUC0-infinity, cumulative amount excreted were calculated for both formulations and subjected to statistical analysis. The relative bioavailability of Formulation 2 with respect to Formulation 1 was 93.76 and 95.31% on the basis of AUC0-24 and cumulative amount excreted, respectively. Statistical comparison of various pharmacokinetic parameters indicated that the two ranitidine tablet formulations are bioequivalent.

Determination of famotidine in low-volume human plasma by normal-phase liquid chromatography/tandem mass spectrometry.

A rapid, sensitive and robust assay procedure using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the determination of famotidine in human plasma and urine is described. Famotidine and the internal standard were isolated from plasma samples by cation-exchange solid-phase extraction with benzenesulfonic acid (SCX) cartridges. The urine assay used direct injection of a diluted urine sample. The chromatographic separation was accomplished by using a BDS Hypersil silica column with a mobile phase of acetonitrile-water containing trifluoroacetic acid. The MS/MS detection of the analytes was set in the positive ionization mode using electrospray ionization for sample introduction. The analyte and internal standard precursor-product ion combinations were monitored in the multiple-reaction monitoring mode. Assay calibration curves were linear in the concentration range 0.5--500 ng ml(-1) and 0.05--50 microg ml(-1) in plasma and urine, respectively. For the plasma assay, a 100 microl sample aliquot was subjected to extraction. To perform the urine assay, a 50 microl sample aliquot was used. The intra-day relative standard deviations at all concentration levels were <10%. The inter-day consistency was assessed by running quality control samples during each daily run. The limit of quantification was 0.5 ng ml(-1) in plasma and 0.05 microg ml(-1) in urine. The methods were utilized to support clinical pharmacokinetic studies in infants aged 0-12 months.

Development and validation of a liquid chromatographic-tandem mass spectrometric method for the determination of pibutidine in human urine.

A liquid chromatographic-tandem mass spectrometric method for the rapid quantitative determination of pibutidine, an H2-receptor antagonist, in human urine has been developed and validated over the concentration range 0.1-25.6 microg ml(-1). Urine samples were prepared based on a simple dilution with 0.05% acetic acid, followed by reversed-phase liquid chromatographic separation. Pibutidine and its internal standard (2H10-pibutidine) were ionized using an electrospray ionization interface and detected by tandem mass spectrometry in the selected reaction-monitoring mode. Completed validation demonstrated the method to be robust, accurate, precise and specific for the direct quantification of pibutidine in human urine. This method has enabled investigation of the urinary excretion of pibutidine following oral administration of pibutidine hydrochloride to healthy subjects.

Automated in-tube solid-phase microextraction-liquid chromatography-electrospray ionization mass spectrometry for the determination of ranitidine.

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was evaluated for the determination of ranitidine. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary column by repeated aspirate/dispense steps. In order to optimize the extraction of ranitidine, several in-tube SPME parameters such as capillary column stationary phase, extraction pH and number and volume of aspirate/dispense steps were investigated. The optimum extraction conditions for ranitidine from aqueous samples were 10 aspirate/dispense steps of 30 microliters of sample in 25 mM Tris-HCl (pH 8.5) with an Omegawax 250 capillary column (60 cm x 0.25 mm I.D., 0.25 micron film thickness). The ranitidine extracted on the capillary column was easily desorbed with methanol, and then transported to the Supelcosil LC-CN column with the mobile phase methanol-2-propanol-5 M ammonium acetate (50:50:1). The ranitidine eluted from the column was determined by ESI-MS in selected ion monitoring mode. In-tube SPME followed by LC-ESI-MS was performed automatically using the HP 1100 autosampler. Each analysis required 16 min, and carryover of ranitidine in this system was below 1%. The calibration curve of ranitidine in the range of 5-1000 ng/ml was linear with a correlation coefficient of 0.9997 (n = 24), and a detection limit at a signal-to-noise ratio of three was ca. 1.4 ng/ml. The within-day and between-day variations in ranitidine analysis were 2.5 and 6.2% (n = 5), respectively. This method was also applied for the analyses of tablet and urine samples.

Rapid characterization of urinary metabolites of pibutidine hydrochloride in humans by liquid chromatography/electrospray ionization tandem mass spectrometry.

The metabolic products of pibutidine hydrochloride, a new H(2)-receptor antagonist, in human urine after oral administration of 40 mg/man were characterized by high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). Two-stage collision-induced dissociation (CID) experiments, with in-source CID by increasing the octapole offset voltage and collision-cell CID, were performed in order to develop a very rapid screening procedure that enhanced selectivity toward pibutidine-related compounds. It was possible to detect metabolites of pibutidine directly from a crude biological matrix without prior extraction, enabling confirmation of the identity of eight metabolites in urine. In addition, the linear range in ESI for pibutidine-related compounds was studied to determine the urinary excretion of pibutidine and its metabolites in humans.

A physiologically based kidney model for the renal clearance of ranitidine and the interaction with cimetidine and probenecid in the dog.

Ranitidine renal clearance was investigated in the beagle dog with or without concomitant infusion of cimetidine or probenecid. Ranitidine was excreted mainly by renal tubular secretion. Plasma clearance was reduced by probenecid from 198 +/- 47 to 119 +/- 41 mL min-1 (mean +/- SD.); renal clearance was reduced from 104 +/- 33 to 54 +/- 24 mL min-1 (p < 0.02) by probenecid and to 89 +/- 37 mL min-1 (NS) by cimetidine. Plasma and urine data were analysed simultaneously with a physiologically based kidney model and were both described adequately by the model, although tubular secretion could not be fully characterized as no saturation was achieved despite high dosages. Tubular secretion of ranitidine was simplified to first-order brush-border and basolateral transport across the proximal tubular cell. Basolateral transport was reduced (from 18.4 +/- 7.8 to 13.6 +/- 10.3 min-1 by cimetidine and 3.9 +/- 3.1 min-1 by probenecid), whereas no effect on brush-border exit was found. Estimated inhibition constants of cimetidine and probenecid were 62 and 4 micrograms mL-1, respectively. Summarizing, ranitidine renal pharmacokinetics were accurately described by the physiologically based kidney model presented in this paper. Model calculations suggest that interaction with cimetidine and probenecid results from competition for basolateral ranitidine uptake into tubular cells.

Pharmacokinetics of intravenous and intragastric cimetidine in horses. I. Effects of intravenous cimetidine on pharmacokinetics of intravenous phenylbutazone.

Cimetidine was administered intravenously and by the intragastric route to six mares at a dose of 4.0 mg/kg of body weight (bw). Specific and sensitive high performance liquid chromatographic methods for the determination of cimetidine in horse plasma and urine and cimetidine sulfoxide in urine are described. Plasma cimetidine concentration vs. time data were analysed by non-linear least squares regression analysis to determine pharmacokinetic parameter estimates. The median (range) plasma clearance (Cl) was 8.20 (4.96-10.2) mL/min.kg of body weight, that of the steady-state volume of distribution (Vdss) was 0.771 (0.521-1.15) L/kg bw, and that of the terminal elimination half-life (t1/2 beta) was 92.4 (70.6-125) minutes. The median (range) renal clearance of cimetidine was 4.08 (2.19-6.23) mL/min.kg bw or 55.4 (36.3-81.8)% of the corresponding plasma clearance. Cimetidine sulfoxide was excreted in urine and its urinary excretion through 8 h accounted for 12.0 (9.8-16.6)% of the plasma clearance of cimetidine. The median (range) extent of intragastric bioavailability was 14.4 (6.82-21.8)% and the maximum plasma concentration after intragastric administration was 0.31 (0.24-0.50) microgram/mL. Intravenous cimetidine had no effect on the disposition of intravenous phenylbutazone or its metabolites except that the maximum plasma concentration of gamma-hydroxyphenylbutazone was less after cimetidine treatment.

Prediction of absolute bioavailability for drugs using oral and renal clearance following a single oral dose: a critical view.

In order to determine the absolute bioavailability, both oral and intravenous administrations of a drug are often used. Recently a new method has been proposed to determine absolute bioavailability in the absence of intravenous dose. Following a single oral dose, this method requires oral and renal clearance data from normal subjects and renal failure patients. The bioavailability is calculated from a plot of oral against renal clearance following an oral dose, where the inverse of the slope is equal to absolute bioavailability. This study examines the prediction of absolute bioavailability from the proposed method for eight drugs which have a wide range of oral and renal clearance. From this study, it appears that the proposed method may not be reliable for the prediction of absolute bioavailability and further investigation is needed to test the validity of this method.

Metabolism of ebrotidine. A review.

A hypothetical metabolic pathway for ebrotidine (N-[(E)-[[(2-[[[2-[(diaminomethylene]amino]-4-thiazolyl] methyl]thio]ethyl]amino]methylene-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) has been proposed on the basis of previous data on the metabolism of other H2-receptor antagonists as well as on in vitro degradation assays of ebrotidine. Its potential metabolites have been synthesized and characterized, and their presence in human urine has been investigated by high-performance liquid chromatography (HPLC). Analytical-scale HPLC allowed the identification of metabolites by means of their retention time and UV spectrum, while semipreparative-scale HPLC allowed their identification through FT-IR and 1H-NMR. Mass spectrometry using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) for HPLC-MS coupling allowed the identification of all metabolites in human urine. The quantitative determination of ebrotidine and its derivatives has been performed according to a newly designed method which consisted of a liquid-liquid extraction in a basic medium followed by reversed-phase HPLC with ion-pair formation. This method was sensitive, precise and no chromatographic interferences with other drugs which might be administered in combination with ebrotidine were observed. In order to elucidate the excretion of ebrotidine, the analytical method was applied to the analysis of the urine collected from 2 healthy volunteers 96 h after receiving 400 mg of ebrotidine.

Pharmacokinetic study of ebrotidine administered in multiple doses to healthy volunteers for 4 days.

The safety of ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4- thiazolyl]methyl]thio]ethyl]amino] methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542), a new H2-receptor antagonist with gastroprotective activity, was assessed and its main pharmacokinetic parameters were determined in order to establish the dose linearity after the repeated administration of three different dose levels. The study was carried out in a group of 8 healthy volunteers of either sex, aged between 20 to 29 years. Oral doses of ebrotidine were administered in a randomized, single-blind design. Volunteers remained in the Unit for two days at each of the three study phases with washout intervals of 2 weeks and received seven doses of ebrotidine (150, 300 and 500 mg b.i.d). Pharmacological evaluation included vital signs, laboratory tests, adverse events and blood and urine samplings for pharmacokinetic analysis. Ebrotidine was determined by high performance liquid chromatography (HPLC) with UV detection. The results showed a good tolerability of ebrotidine after the administration of seven doses for 4 days, with no changes in the vital signs or laboratory parameters. No clinically significant dose-related adverse events were reported during the study. The absorption of ebrotidine was relatively rapid (tmax approximately 2 h) and linear within the dose range from 150 to 500 mg. Drug biotransformation was linear with doses tested, and no metabolic saturation occurred. The terminal elimination half-life of ebrotidine was between 7 and 11 h or even longer. There was no accumulation of ebrotidine and the steady state was reached, regardless of the dose administered, within the first 24-48 h.

Pharmacokinetics of ebrotidine in healthy volunteers. A summary.

Several clinical pharmacokinetic studies of ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl] methyl]thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) administered by oral route in single and multiple doses to healthy volunteers have been performed. Dosage levels were 150, 300, 400, 500, 600 and 800 mg. Plasma concentrations of unchanged ebrotidine and its major metabolite, ebrotidine sulfoxide, excreted in the urine were determined. The main pharmacokinetic parameters were calculated from the experimental data. Absorption was relatively rapid (Imax = 2 h) and unrelated to dose. Drug behavior was considered as reasonably linear: Cmax = 364-1168 ng/ml and AUC0-12 h = 1427-5997 ng.h/ml (doses from 150 mg to 800 mg). The mean values of terminal elimination half-life (t1/2 beta) ranged from 13.9 to 20.3 h (doses of 400, 600 and 800 mg). After multiple dosing there was no drug accumulation, and no significant changes in the mean values of the main pharmacokinetic parameters were observed. The steady state was reached from the second day of administration, 10-24% of the ebrotidine administered dose was excreted in urine mainly as its major metabolite, ebrotidine sulfoxide, as well as unchanged drug and other minor metabolites. These percentages were constant and independent of the dose administered.