Venkata K. Boppana

Use of a post-column immobilized β-glucuronidase enzyme reactor for the determination of diastereomeric glucuronides of fenoldopam in plasma and urin

Abstract A post-column enzyme reactor, containing β-glucuronidase immobilized on controlled-pore glass beads, was developed for use in the high-performance li but the efficiency gradually declined thereafter until, at 50% methanol, the reactor was inactive. The working pH range for the mobile phase was 5.5–

High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine.

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.

Simplified procedures for the determination of fenoldopam and its metabolites in human plasma by high-performance liquid chromatography with electrochemical detection: comparison of manual and robotic sample preparation methods

Quantitative analytical methods, based on high-performance liquid chromatography with electrochemical detection, were developed for fenoldopam and its metabolites in human plasma. Two extraction methods, a liquid-liquid extraction method for fenoldopam and its methoxy metabolites and a liquid-solid extraction procedure for the sulfate and glucuronide conjugates of fenoldopam were developed. The extractions can either be performed manually or by robot. The limit of detection for fenoldopam, its sulfate and methoxy metabolites was 0.025, 2 and 0.5 ng/ml, respectively, at a signal to noise ratio of 4. The intra-assay and inter-assay coefficients of variation for both manual and robotic extraction procedures were comparable. These methods were suitably selective and sensitive for pharmacokinetic and metabolic studies of fenoldopam.

High-performance liquid chromatographic determination of guanidino compounds by automated pre-column fluorescence derivatization

Abstract An automated pre-column derivatization approach was used to develop an high-performance liquid chromatographic method for the determination of endogenous guanidino compounds. A commercially available autoinjector which was capable of adding and mixing reagent solutions and timing the reaction prior to injection was used to generate a fluorescent product by reaction of guanidino compounds with alkaline ninhydrin. The fluorescent products were separated by reversed-phase chromatography and detected with a fluorometer. Optimization of the pre-column reaction conditions resulted in a simple, highly sensitive and specific analytical method for the determination of guanidino compounds with excellent reproducibility and linearity. Application of this methodology to the determination of methylguanidine in human plasma samples resulted in a limit of quantification of 1 ng/ml (13.7 pmol/ml). The method was successfully employed for the quantification of circulating levels of methylguanidine in normal human subjects and uremic patients. The methodology should be generally applicable to the detection of other guanidino compounds in biological fluids.

Determination of fenoldopam (SK&F 82526) and its metabolites in human plasma and urine by high-performance liquid chromatography with electrochemical detection

Fenoldopam [6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-di ol] is a potent renal vasodilator that is currently undergoing Phase II clinical trials. Quantitative analytical methods, based on high-performance liquid chromatography with electrochemical detection (HPLC-ED) after ethyl acetate extraction from plasma or urine were developed for the determination of fenoldopam and its identified metabolites in biological media. The lower limit of quantitation for fenoldopam in plasma was 50 pg/ml. In assays for fenoldopam glucuronide(s) and fenoldopam conjugates, urine was treated with beta-glucuronidase and Glusulase, respectively, and the liberated fenoldopam was quantified by HPLC-ED. A novel assay by dual-electrode (in series) HPLC-ED was developed for the 8-sulfate of fenoldopam. In this method, the 8-sulfate was oxidized to the o-quinone at the first electrode and quantitated at the second electrode after reduction to the catechol. A similar dual-electrode HPLC-ED method was used for 7- and 8-O-methyl fenoldopam. Conjugates of the O-methyl metabolites were determined by HPLC-ED after hydrolysis to O-methyl fenoldopam. These methods have been used to study the kinetics and metabolism of fenoldopam in healthy volunteers. The methods are specific, sensitive, reproducible, and linear over a wide range of concentrations. Precision of the analyses, expressed as coefficients of variation, were less than 10% for all analyses.

High-performance liquid chromatographic determination of an arginine-containing octapeptide antagonist of vasopressin in human plasma by means of a selective post-column reaction with fluorescence detection

A novel high-performance liquid chromatographic method was developed for the determination in plasma samples of the synthetic octapeptide SK&F 105494 (O-ethyl-D-tyrosyl-L-phenylalanyl-L-valyl-L-asparaginyl-5-[1- (carboxymethyl)cyclohexyl]-L-norvalyl-L-arginyl-D-arginamide , cyclic (5-1) peptide), an active aquaretic agent which significantly increases water excretion in experimental animal models through competitive antagonism of renal epithelial vasopressin receptors. The method involves isolation of Sk&F 105494 from plasma samples by solid phase extraction prior to chromatographic analysis. Following chromatographic separation, in-line fluorescence detection was accomplished via a selective-post-column reaction of the guanidino group of the arginine moiety of the peptide with alkaline ninhydrin to generate a highly fluorescent product. Optimization of the post-column reaction conditions and the use of high-performance liquid chromatography columns of reduced internal diameter (2.1 mm), resulted in an on-column detection limit of 50 pg (45 fmol). The limit of sensitivity in plasma was 0.5 ng/ml. The assay was linear over the range 0.5-100 ng/ml. Precision and accuracy were within 11% across the calibration range. The assay was shown to be suitable to study the pharmacokinetics of SK&F 105494 in human subjects and animals. In addition, the methodology developed has general applicability in the detection of arginine-containing peptides in biological matrices.

The effect of acetaminophen on the disposition of fenoldopam: Competition for sulfation

Both fenoldopam and acetaminophen undergo conjugation with sulfate in humans. The present study was undertaken to determine if a metabolic interaction exists between these two compounds in humans. Twelve healthy male volunteers participated in a single‐dose crossover study with 100 mg fenoldopam (capsule) alone or in combination with 1000 mg acetaminophen. The effects of chronic acetaminophen dosing (1000 mg three times/day for 7 days) on a single 100 mg tablet of fenoldopam were studied in a second crossover study in seven additional volunteers. Concomitant dosing with fenoldopam with acetaminophen resulted in increases in peak fenoldopam plasma concentration and AUC after both single (+32% and +50%, respectively) and chronic (+73% and +66%, respectively) acetaminophen dosing. Decreases in peak plasma concentrations and AUC for fenoldopams sulfated metabolites were seen in both studies. These findings indicate a metabolic basis for the interaction between fenoldopam and acetaminophen, presumably through a competition for inorganic sulfate.

Interspecies Pharmacokinetics of a Novel Hematoregulatory Peptide (SK&F 107647) in Rats, Dogs, and Oncologic Patients

AbstractPurpose. To study the pharmacokinetics of SK&F 107647, a novel hematoregulatory agent, in rats, dogs, and patients with non-lymphoid solid tumor malignancy. Methods. Sprague Dawley rats and beagle dogs (n = 6 each; 3 M, 3 F) were given 25 mg/kg of SK&F 107467 as an iv bolus injection, and patients (n = 6; 4 M, 2 F) received 100 ng/kg as a 2 hour iv infusion. Plasma samples were assayed for drug using either HPLC (rat and dog) or RIA (human). Results. In each species the plasma clearance (CL) of SK&F 107647 was low in relation to hepatic blood flow, and the volume of distribution (Vdss) was reflective of distribution to extracellular body water. The plasma CL in humans was near that of average glomerular filtration rate. Using allometric equations for interspecies scaling (Y = a·Wb), body-weight normalized human pharmacokinetic data were reasonably predicted using either the body weight normalized rat or the dog data. The allometric exponents (b) for CL, Vdss, and T1/2 of SK&F 107647 were 0.63, 0.94, and 0.29, respectively. Conclusions. Use of a limited pool of available animal data allowed for reasonable predictions of human pharmacokinetics of SK&F 107647.

A Retrospective Analysis of Fenoldopam Renal Excretion in 65 Subjects: Evidence for Possible Intrarenal Formation of Fenoldopam from Its Metabolites

Clinical studies have suggested that the dopamine DA1 agonist, fenoldopam, may exhibit nonlinear renal excretion in humans. A retrospective population pharmacokinetic analysis of the renal excretion of fenoldopam and one of its major metabolites, fenoldopam-8-sulfate, was conducted in 65 healthy volunteers to examine this phenomenon. Fenoldopam-8-sulfate exhibited a mean (±SE) renal plasma clearance of 129 ± 4 ml/min, which was independent of its AUC. In contrast, fenoldopam renal plasma clearance ranged from 2220 to 150 ml/min and decreased nonlinearily with increasing fenoldopam AUC. Fenoldopam renal clearance was characterized as a function of fenoldopam AUC using a nonlinear saturation model. The analysis predicted an initial maximal renal clearance of 2852 ml/min, which decreased to 78 ml/min at maximal inhibition. The fenoldopam AUC required to half-saturate fenoldopam renal clearance was 5.2 ng × hr/ml. The elevated clearance values for fenoldopam, beyond normal physiologic limits for renal blood flow in man, suggest that intrarenal formation of fenoldopam from one or more of its circulating metabolites may be contributing to the observed nonlinear decreases in fenoldopam renal excretion. Preliminary data from our laboratory suggest that in vivo desulfation of fenoldopam-8-sulfate to fenoldopam does occur in the dog.

Determination of oxiracetam in human plasma by reversed-phase high-performance liquid chromatography with fluorimetric detection

Reversed-phase HPLC methodology utilizing pre-column derivatization and post-column reaction fluorimetric detection has been developed and applied to the determination of oxiracetam in human plasma. The method involves preliminary isolation of oxiracetam and internal standard from plasma by solid-phase extraction prior to the formation of their n-propyl carbamate derivatives. The carbamate derivatives were subsequently isolated by solid-phase extraction and subjected to a gradient liquid chromatographic separation on an octadecylsilica column prior to on-line post-column alkaline hydrolysis to produce the corresponding primary amine, which was in turn derivatized with o-phthalaldehyde and 3-mercaptopropionic acid to yield a fluorescent isoindole. The isoindole was then quantified using a fluorescence detector. The method provided an on-column detection limit of 0.5 ng of oxiracetam and was sufficiently sensitive, accurate, and precise to support pre-clinical or clinical pharmacokinetic studies.