Simultaneous determination of rabeprazole and its two active metabolites in human urine by liquid chromatography with tandem mass spectrometry and its application in a urinary excretion study.

A simple and rapid liquid chromatography with tandem mass spectrometry method has been developed and validated for the determination of rabeprazole and its two active metabolites, rabeprazole thioether and desmethyl rabeprazole thioether, in human urine using donepezil as the internal standard. The sample preparation procedure involved a simple dilution of urine sample with methanol (1:3, v/v). The chromatographic separation was achieved on a Hedera ODS-2 C18 column using a mixture of methanol/10 mmol/L ammonium acetate solution (containing 0.05% formic acid; 55:45, v/v) as the mobile phase. The method was validated over the concentration ranges of 0.15-100 ng/mL for rabeprazole, 0.30-400 ng/mL for rabeprazole thioether, and 0.05-100 ng/mL for desmethyl rabeprazole thioether. The established method was highly sensitive with a lower limit of quantification of 0.15 ng/mL for rabeprazole, 0.30 ng/mL for rabeprazole thioether, and 0.05 ng/mL for desmethyl rabeprazole thioether. The intra- and interbatch precision was <4.5% for the low, medium, and high quality control samples of all the analytes. The recovery of the analytes was in the range 95.4-99.0%. The method was successfully applied to a urinary excretion profiles after intravenous infusion administration of 20 mg rabeprazole sodium in healthy volunteers.

Modified Au nanoparticles-imprinted sol-gel, multiwall carbon nanotubes pencil graphite electrode used as a sensor for ranitidine determination.

A new, simple, and disposable molecularly imprinted electrochemical sensor for the determination of ranitidine was developed on pencil graphite electrode (PGE) via cyclic voltammetry (CV). The PGEs were coated with MWCNTs containing the carboxylic functional group (f-MWCNTs), imprinted with sol-gel and Au nanoparticle (AuNPs) layers (AuNP/MIP-sol-gel/f-MWCNT/PGE), respectively, to enhance the electrode's electrical transmission and sensitivity. The thin film of molecularly imprinted sol-gel polymers with specific binding sites for ranitidine was cast on modified PGE by electrochemical deposition. The AuNP/MIP-sol-gel/f-MWCNT/PGE thus developed was characterized by electrochemical impedance spectroscopy (EIS) and CV. The interaction between the imprinted sensor and the target molecule was also observed on the electrode by measuring the current response of 5.0mMK3[Fe(CN)6] solution as an electrochemical probe. The pick currents of ranitidine increased linearly with concentration in the ranges of 0.05 to 2.0µM, with a detection limit of (S/N=3) 0.02µM. Finally, the modified electrode was successfully employed to determine ranitidine in human urine samples.

Identification of new omeprazole metabolites in wastewaters and surface waters.

Omeprazole is one of the world-wide most consumed pharmaceuticals for treatment of gastric diseases. As opposed to other frequently used pharmaceuticals, omeprazole is scarcely detected in urban wastewaters and environmental waters. This was corroborated in a previous research, where parent omeprazole was not detected while four transformation products (TPs), mainly resulting from hydrolysis, were found in effluent wastewaters and surface waters. However, the low abundance of omeprazole TPs in the water samples together with the fact that omeprazole suffers an extensive metabolism, with a wide range of excretion rates (between 0.01 and 30%), suggests that human urinary metabolites should be investigated in the water environment. In this work, the results obtained in excretion tests after administration of a 40 mg omeprazole dose in three healthy volunteers are reported. Analysis by liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF MS) reported low concentrations of omeprazole in urine. Up to twenty-four omeprazole metabolites (OMs) were detected and tentatively elucidated. The most relevant OM was an omeprazole isomer, which obviously presented the same exact mass (m/z 346.1225), but also shared a major common fragment at m/z 198.0589. Subsequent analyses of surface water and effluent wastewater samples by both LC-QTOF MS and LC-MS/MS with triple quadrupole revealed that this metabolite (named as OM10) was the compound most frequently detected in water samples, followed by OM14a and OM14b. Up to our knowledge, OM10 had not been used before as urinary biomarker of omeprazole in waters. On the contrary, parent omeprazole was never detected in any of the water samples. After this research, it seems clear that monitoring the presence of omeprazole in the aquatic environment should be focused on the OMs suggested in this article instead of the parent compound.

Pharmacokinetic and pharmacodynamic alterations in the Roux-en-Y gastric bypass recipients.

OBJECTIVE: We conducted a pharmacokinetic (PK) study and a pharmacodynamic (PD) study to assess whether Roux-en-Y gastric bypass (RYGB) surgery is associated with significant changes to PK and PD of oral medications. BACKGROUND: The effect of RYGB on oral drug disposition is not well understood. METHODS: An oral cocktail of probe drugs for major drug-metabolizing enzymes (caffeine, tolbutamide, omeprazole, dextromethorphan, and oral and intravenous midazolam) was administered to 18 RYGB recipients and 18 controls. Timed blood and urine samples were obtained for PK analyses. Forty mg of oral furosemide was administered to 13 RYGB recipients and 14 controls, and urine and blood samples were collected for assessing furosemidePK, and urine volume and urine sodium excretion for PD analyses. RESULTS: Compared with controls, the RYGB group had significantly lower time to maximum plasma concentration (tmax) for caffeine (0.58 ± 0.5 vs 2.1 ± 2.2 hours, P < 0.0001), tolbutamide (1.4 ± 1.8 vs 2.1 ± 2.2 hours, P = 0.0001), omeprazole (1.1 ± 1.1 vs 4.4 ± 1.3 hours, P < 0.0001), and oral midazolam (0.5 ± 0.2 vs 0.7 ± 0.4 hours, P < 0.01). However, maximum plasma concentration, half-life, area under the curve, and oral bioavailability were not different. Compared with controls, the RYGB group had brisk natriuresis, with significantly lower tmax for urine sodium (1.3 ± 0.5 vs 3.1 ± 2.3 hours, P < 0.02) and correspondingly lower tmax for furosemide (1.8 ± 0.3 vs 4.2 ± 1.2 hours, P = 0.006). However, 6-hour urine sodium and 6-hour urine volume were not different between the two groups. CONCLUSIONS: RYGB recipients have significantly shorter tmax for the studied orally administered medications, but otherwise no other significant changes in PK were reported.

Analysis of omeprazole and its main metabolites by liquid chromatography using hybrid micellar mobile phases.

Omeprazole is a selective inhibitor of gastric acid secretion and is one of the most widely prescribed drugs internationally. A chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulphate and propanol buffered at pH 7 and a C18 column is reported for the determination of omeprazole and its principal metabolites (omeprazole sulphone and hydroxyomeprazole) in urine and serum samples. In this work, direct injection and UV detection set at 305 nm was used. Omeprazole and its metabolites were eluted in less than 11min with no interference by the protein band or endogenous compounds. Adequate resolution was obtained with a chemometric approach, in which the retention factor and shape of the chromatographic peaks were taken into account. The analytical parameters including linearity (r>0.9998), intra- and inter-day precision (RSD, %: 0.6-7.9 and 0.14-4.7, respectively) and robustness were studied in the validation of the method for the three compounds. The limits of detection and quantification were less than 6 and 25ngmL(-1), respectively. Recoveries in micellar medium, plasma and urine matrices were in the 98-102% range. Finally, the method was successfully applied to the determination of omeprazole and its metabolites in physiological samples. Omeprazole was also analysed in pharmaceutical formulations.

Validation of a simplified method for determination of cimetidine in human plasma and urine by liquid chromatography with ultraviolet detection.

A HPLC method was developed for determination of cimetidine in human plasma and urine. Plasma samples were alkalinized followed by liquid extraction with water-saturated ethyl acetate then evaporated under nitrogen. The extracts were reconstituted in mobile phase and injected onto a C(18) reversed-phase column; UV detection was set at 228 nm. Urine samples were diluted with an internal standard/mobile phase mixture (1:9) prior to injection. The lower limit of quantification in plasma and urine were 100 ng/ml and 10 microg/ml, respectively; intra- and inter-day coefficients of variation were

Positive predictive values of abused drug immunoassays on the Beckman Synchron in a veteran population.

The pressure to reduce the cost of analytic testing makes it tempting to discontinue routine confirmation of urine specimens positive for drugs of abuse by immunoassay. Beyond the economic motivation, the requirement for confirmation should be driven by the positive predictive value of the screening tests. We have quantitated positive predictive values of our screening immunoassays in a large metropolitan Veterans Affairs Medical Center. We reviewed the confirmatory rate of urine specimens positive for drugs of abuse with Beckman Synchron reagents from June 1998 to June 1999 and tabulated the false-positive screening rate. There were 175 instances of false-positive screens during the 13 months we analyzed. Positive predictive values ranged from 0% (amphetamine) to 100% (THC). We determined that the low positive predictive value of the amphetamine assay in our laboratory was primarily due to the use of ranitidine (Zantac). Urine specimens containing greater than 43 microg/mL ranitidine were positive in our amphetamine assay. This concentration is routinely exceeded in our patients taking ranitidine. In our clinical and analytic setting, the Beckman THC assay did not require confirmation. The positive predictive values of the Beckman opiate, cocaine, barbiturate, propoxyphene, and methadone immunoassays dictate routine confirmatory testing in specimens that screen positive for these substances. Finally, because of its extreme sensitivity to ranitidine, the Beckman amphetamine assay has little utility in our laboratory setting.

Sex-related differences in urinary excretion of egualen sodium in rats.

Egualen sodium (sodium 3-ethyl-7-isopropyl-1-azulenesulfonate 1/3 hydrate) is a new antiulcer drug. There has been no difference observed in absorption between male and female rats, the relative amount of metabolites in male plasma has been higher than that in females, and the excretion ratios of metabolites in males have been significantly higher than those in females. However, the plasma concentration profile of total radioactivity in males has been higher than that in females. To clarify this discrepancy, the renal clearances and plasma concentrations of the unchanged drug and its metabolites were determined. The renal clearance of the unchanged drug in males was 21 times lower than that in females, and the urinary excretions in males and females were 2.1 and 39.5% of dose, respectively. This indicates that the major factor in the sex-related difference observed in the plasma concentration of total radioactivity is due to the difference in the renal clearance of the unchanged drug between the sexes. The results of treatments with probenecid in normal and gonadectomized rats revealed that egualen sodium was mainly excreted into urine by secretion through the renal tubule. Furthermore, the results of treatments with testosterone in rats revealed that the excretion of egualen sodium was highly affected by androgens. These facts indicated that the sex-related difference observed in the plasma concentration of total radioactivity can be attributed to the inhibition of renal tubular secretion of the unchanged drug by androgens. This is the first example of sex-related differences in both metabolism and excretion.

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.

A method for measuring naringenin in biological fluids and its disposition from grapefruit juice by man.

The major flavonoid in grapefruit juice, naringin, has an aglycone, naringenin, that inhibits some oxidations in vitro and may have in vivo activity. We developed an HPLC method to measure naringenin using a methanol:water mobile phase with UV absorbance detection. The recovery of naringenin was 96%. Two subjects who drank grapefruit juice containing 214 mg naringin daily excreted approximately 30 mg/day of naringenin glucuronide. Thus, naringin is hydrolyzed to naringenin and then conjugated with glucuronic acid prior to excretion of the conjugate.

Determination of a new antiulcer agent, eupatilin, in rat plasma, bile, urine, and liver homogenate by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a new antiulcer agent, eupatilin, in rat plasma, urine, bile, and liver homogenate. The method involved deproteinization of biological sample with the same volume of acetonitrile. A 100 microl aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase employed was ammonium acetate buffer (1% ammonium acetate and 0.5% acetic acid) - acetonitrile (58:42, v/v) and the flow rate was 1.0 ml/min. The column effluent was monitored by a ultraviolet detector set at 350 nm. The retention time for eupatilin was approximately 6.5 min. The detection limits for eupatilin in rat plasma, urine, bile, and liver homogenate were 50, 50, 100, and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low (below 7.46%) for rat plasma, urine, bile, and liver homogenate. No interferences from endogenous substances were observed.

Determination of a new antiulcer agent, YJA-20379-1 in plasma, urine, blood, and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a new antiulcer agent, YJA-20379-1, in human plasma and urine, and rat blood and tissue homogenates. The sample preparation was simple: a 2.5-volume of acetonitrile was added to the biological sample to deproteinize it. A 50-microliter aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase employed was methanol--0.1 M Sørensen phosphate buffer of pH7.0--H2O (80:2:15, v/v/v), and was run at a flow-rate of 0.7 ml/min. The column effluent was monitored by ultraviolet detector at 254 nm. The retention time for YJA-20379-1 was approximately 6.3 min. The detection limits for YJA-20379-1 in human plasma and urine, and rat tissue homogenate (including blood) were 50, 100, and 100 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 9.44%) for the human plasma and urine, and rat blood and tissue homogenate. No interference from endogenous substances were found.

Use of post-column fluorescence derivatization to develop a liquid chromatographic assay for ranitidine and its metabolites in biological fluids.

Ranitidine and its main metabolites, ranitidine N-oxide and ranitidine S-oxide, were determined in plasma and urine after separation using reversed-phase liquid chromatography. The mobile phase consisted of an initial isocratic step with 7:93 (v/v) acetonitrile-7.5 mM phosphate buffer (pH 6) for 8 min, followed by a linear gradient up to a 25:75 (v/v) mixture over 1 min. Detection was carried out by a post-column fluorimetric derivatization based on the reaction of the drugs with sodium hypochlorite, giving rise to primary amines that reacted with o-phthalaldehyde and 2-mercaptoethanol to form highly fluorescent products. The calibration graphs, based on peak area, were linear in the range 0.1-4 microg/ml for all drugs. The detection limits were 30, 41 and 32 ng/ml (8.6, 12.5 and 9.1 pmol) for ranitidine S-oxide, ranitidine N-oxide and ranitidine, respectively. Chromatographic profiles obtained for plasma and urine samples showed no interference from endogenous compounds.

Saturable urinary excretion kinetics of famotidine in the dog.

An important elimination route of the histamine H2 antagonist famotidine is active tubular secretion via the renal organic cation transport system. To characterize the excretion kinetics of famotidine in-vivo, the relationship between plasma concentration and urinary excretion rate was investigated in the beagle dog over a wide concentration range. The maximum transport capacity and the apparent Michaelis-Menten constant of tubular secretion were estimated. Concentration-dependent renal clearance was determined either after intravenous infusion of high doses of famotidine for a short time or during continuous infusion. From individual experiments only indications of saturation were observed; these could not be quantified. A tubular titration curve, in which the active tubular famotidine secretion was plotted against the plasma concentration, was constructed from the data from all the experiments. Active tubular secretion was calculated for each experiment separately by subtracting the famotidine filtration rate from the total excretion rate. A tubular transport maximum of 2400 +/- 220 micrograms min-1 and an apparent Michaelis-Menten constant for tubular secretion of 26 +/- 4 micrograms mL-1 (76 +/- 12 microM) were estimated from the curve. To the best of our knowledge, this is the first time that saturation of famotidine renal clearance has been fully quantified in-vivo. Considering the low therapeutic plasma concentrations of famotidine (< 0.1 microgram mL-1), these results suggest that clinically the drug has a low interactive potential.

Determination of ebrotidine and its metabolites in human urine by reversed-phase ion-pair high-performance liquid chromatography.

Ebrotidine is a new H2-receptor antagonist with powerful antisecretory activity, demonstrated gastroprotection and the ability to inhibit protease and lipase activities of Helicobacter pylori. As a tool in the clinical pharmacokinetic study of ebrotidine, an analytical method for the simultaneous determination of ebrotidine an its metabolites in human urine was developed. An ion-pair reversed-phase HPLC separation using 1-hexanesulfonic acid and acetonitrile as mobile phase with gradient elution was optimized. In addition, several procedures of preconcentration and clean-up were tested, including solid-phase and liquid-liquid extraction, the mixture dichloromethane-2-propanol (9:1, v/v) at pH 11 being the most efficient. The quality parameters of the whole analytical method were established, the calibration curves were linear over the range studied (1-200 micrograms/ml) and the reproducibility of the method was high (inter-day R.S.D. values lower than 4.4%). The limits of detection were between 26 and 110 ng/ml of urine for ebrotidine and its metabolites. The method was applied to the analysis of urine collected from two volunteers during 96 h following oral administration of ebrotidine at a dose of 400 mg.

Mass balance of 14C-bismuth sucrose octasulfate in Sprague-Dawley rats: evidence for dissociation of bismuth from sucrose octasulfate.

The mass balance of 14C bismuth sucrose octasulfate (BISOS) was investigated in eight male Sprague-Dawley rats after single oral doses of 1.0 g kg-1. Bismuth and radioactivity were monitored in blood, urine, and feces for up to 144 h post-dose, while kidneys, brain, liver, and lungs were assayed for bismuth at 144 h post-dose. In a separate experiment, bismuth was monitored in bile of bile-duct-cannulated animals for 48 h post-dose. Fecal excretion of bismuth averaged 95.8 +/- 5.30% bismuth dose, while 99.2 +/- 3.63% of the radiolabel was excreted in feces. Urinary excretion of bismuth averaged 0.051 +/- 0.028% bismuth dose, and 1.83 +/- 1.08% radioactive dose. Biliary excretion of bismuth averaged 0.0003 +/- 0.0006% bismuth dose, and 0.026 +/- 0.030% radiolabeled dose. An average 0.005 +/- 0.002% of the bismuth dose was present in kidney, liver, and lung. Bismuth levels in brain were below quantifiable limits. Though BISOS contains 57.3% by weight of bismuth, peak blood concentrations of bismuth were three orders of magnitude lower than for BISOS equivalents (Cmax for BISOS averaged 110 +/- 55.4 micrograms eq mL-1, while for bismuth it was 26.1 +/- 10.3 ng mL-1). This data indicates that bismuth dissociates from sucrose octasulfate, probably during the absorption phase, and exhibits differential pharmacokinetic characteristics from sucrose octasulfate. The low biliary and urinary excretion of both bismuth and BISOS equivalents is indicative of low systemic absorption. Greater than 96% recovery in feces, bile, and urine indicates that mass balance was achieved following oral administration.

Determination of a new antiulcer agent, YJA-20379-2, in human plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a new antiulcer agent, YJA-20379-2, in human plasma and urine. The sample preparation was simple: 2.5-volume of acetonitrile was added to the biological sample to deproteinize. A 50-microliter aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase employed was methanol-0.1M Sørensen phosphate buffer of pH 7.0-H2O (75:2:25, v/v/v), and was run at a flow-rate of 1.0 ml/min. The column effluent was monitored by ultraviolet detector at 295 nm. The retention time for YJA-20379-2 was approximately 7.0 min. The detection limits for YJA-20379-2 in human plasma and urine were both 100 ng/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 9.16%) for both the human plasma and urine. No interference from endogenous substances was found.

Pharmacokinetics and tolerance of pantoprazole, a proton pump inhibitor after single and multiple oral doses in healthy Japanese volunteers.

The pharmacokinetics and tolerance of pantoprazole were investigated after single (20, 40, 80, and 120 mg) and multiple (80 mg once a day for 7 days) oral administration as enteric-coated tablet formulation to healthy male Japanese volunteers. Pantoprazole was well tolerated with no serious adverse events at all doses. Pantoprazole was rapidly absorbed in the fasted state. The mean maximum concentration in serum (Cmax) ranged from 1.77-9.25 micrograms/ml for the 20-120 mg dose and the mean time to reach Cmax (tmax) ranged from 1.92-2.42 h. The half-life (t1/2) ranged from 0.74-1.16 h. A good linear correlation was found between the administered doses (20-120 mg) and the resulting area under the concentration-time curve (AUC) and Cmax with the correlation coefficients of 0.9088 and 0.9263, respectively. Within 24 h, pantoprazole was excreted into urine as the unchanged drug to a negligible extent. In the multiple dose study, 2 apparent poor metabolizers (PMs) of pantoprazole were observed. The means of Cmax, AUC and t1/2 for these 2 PMs were 1.6, 6.7, and 6.8 times higher than those of the extensive metabolizers (EMs). The pharmacokinetic parameters such as Cmax, AUC, and t1/2 after the 7th oral dose were not significantly different from those after the 1st dose both in the PMs and the EMs, which indicated that there was virtually no drug accumulation.

Lack of pharmacokinetic interaction between butorphanol nasal spray and cimetidine.

The potential for a pharmacokinetic interaction between butorphanol nasal spray and cimetidine, under steady state conditions, was evaluated in 16 healthy male volunteers. Subjects received either a 1 mg butorphanol nasal spray every 6 h or a 300 mg cimetidine tablet every 6 h on days 1 4, the combination of two compounds every 6 h on days 5-8 and the original treatment as described in the first segment (days 1-4) on days 9-12. Serial blood and urine samples were collected on days 4, 8 and 12, and additional blood samples were taken immediately, prior to the morning dose on days 3, 7 and 11. Based on the analysis of the Cmin samples, the plasma concentrations of cimetidine and butorphanol achieved steady state by the third day of dosing. No statistically significant differences were found in the plasma concentrations of butorphanol or cimetidine (except for t1/2 and MRT) between any of the treatment phases. Butorphanol nasal spray and cimetidine can be co-administered without any adjustment of dosage for either drug.

Identification of major biliary and urinary metabolites of ecabapide in rats.

1. The structures of major biliary and urinary metabolites of ecabapide in rat were identified by comparison with authentic standards using lc-ms and 1H-nmr spectrometry. 2. A major metabolite was found in the bile obtained from rat after an oral dose of 14C-ecabapide and identified as the amidaldehyde derivative. In the urine, two polar metabolites were characterized as the phenolic sulphates. Further, two lipophilic metabolites were identified as alcohol derivatives, and two others as oxamic acids. 3. From these results, it was estimated that the first step in the metabolism of ecabapide in rat was oxidative N-dealkylation to produce the amidaldehyde. This amidaldehyde was further metabolized by two routes, one by reduction of the amidaldehyde into the corresponding alcohol followed by mono-demethylation and subsequent aromatic O-sulphation, the second by oxidation of the amidaldehyde into the oxamic acid followed by mono-demethylation and subsequent aromatic O-sulphation.