Stereospecific determinations of (+/- )-DU-124884 and its metabolites (+/- )-KC-9048 in human plasma by liquid chromatography.

(+)-DU-124884 is a 5-HT1-like receptor agonist under investigation for drug development. A sensitive, stereospecific LC method was developed for the analysis of (+)-DU-124884, its optical isomer (-)-DU-124884 and their N-dealkylated metabolites, (+/- )-KC-9048, in human plasma. A plasma sample was treated with triethylamine in methanol and the proteins were precipitated by acetonitrile. The supernatant was evaporated to dryness under nitrogen. The analytes and internal standard (acebutolol) formed diastereomers with (S)-(+)-1-(1-naphthyl)ethyl isocyanate immediately. The diastereomers formed were extracted into diethyl ether. They were completely resolved from each other and from matrix peaks on a Microsorb silica column with a mobile phase of methanol-chloroform-hexane (8:12:80, v/v/v) in a run time of 26 min. Detection was by fluorescence with excitation wavelength at 320 nm and emission wavelength at 440 nm. The linearity range is 0.1-200 ng ml-1 (r > 0.99). The limit of quantitation is 0.1 ng ml-1 and the detection limit is 0.02 ng ml-1 (signal-to-noise ratio = 3). The interday precision and accuracy of quality control samples were 5.5-7.6% RSD (relative standard deviation) and 0 to+4% bias for (+)-DU-124884, 5.5-7.9% RSD and 0 to +4% bias for (-)-DU-124884, 4.5-6.5% RSD and -7 to 0% bias for (+)-KC-9048 and 4.5-7.5% RSD and -7 to 0% bias for (-)-KC-9048. Consistent recovery from different lots of human plasma, parallelism of the method, stabilities of on-system, reinjection, bench-top, freeze-thaw cycles and sample storage were established.

Direct determination of substituted azepinoindole enantiomers in rat plasma using silica stationary phase and beta-cyclodextrin as a mobile phase additive.

DU 124884 is a racemic serotonin receptor agonist in an early stage of drug development. DU 124884 and its potential N-desmethyl metabolite, KC 9048, both contain a single chiral center. A direct enantioselective HPLC assay was developed and validated to quantify DU 124884 and KC 9048 in rat plasma. The drug and metabolite enantiomers were extracted from plasma and separated using silica stationary phase with an aqueous mobile phase containing beta-cyclodextrin (beta-CD), triethylamine, and 2-methyl-2-propanol. A variable wavelength detector was used to monitor absorbance at 231 nm. The assay calibration range was from 100 to 5000 ng/mL. Quality control sample precision (< or = 9% RSD) and accuracy (+/-10% error) were satisfactory for all four analytes (n = 12). The method was used to assess drug exposure during a pilot toxicology study in rats. Toxicokinetic study animals were dosed subcutaneously for 15 days at 0, 2.5, 10, and 40 mg of DU 124884.HCl kg-1 day-1. Blood was collected on the last day of dosing between 22 min and 4 h and 13 min after the last dose. The samples showed (+/-)-DU 124884 isomer ratios ranging from 1.1 to 1.3. These data suggest that DU 124884 undergoes stereoselective metabolism in rats. Levels of the N-desmethyl metabolite enantiomers were < 100 ng/mL.

Determination of fluvoxamine in human plasma by high-performance liquid chromatography with fluorescence detection.

Fluvoxamine and nortriptyline, the assay internal standard, were extracted from plasma with ethyl acetate, then reacted with dansyl chloride. The derivatives were quantitated by isocratic reversed-phase high-performance liquid chromatography with fluorescence detection. The assay calibration range for fluvoxamine was 10-1000 ng/ml using a 1-ml plasma sample. Pooled plasma quality control sample relative recoveries at 25 and 250 ng/ml were 103 and 105%, respectively. Estimates of quality control inter-day precision during validation were less than or equal to 3% relative standard deviation. The assay was cross-validated with a gas chromatographic method and has been employed in therapeutic drug level monitoring.

Determination of an antipsychotic agent (ICI 204,636) and its 7-hydroxy metabolite in human plasma by high-performance liquid chromatography and gas chromatography-mass spectrometry.

ICI 204,636 (I) is an orally active antipsychotic agent under development for the treatment of schizophrenia in humans. It is partially converted in animals to an active 7-hydroxy metabolite (II). Methods were developed for the simultaneous determination of both analytes in human plasma using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The analytes were extracted from plasma using phenyl solid-phase extraction columns. Quantification by isocratic HPLC was performed in the reversed-phase mode with detection at 250 nm. Extracts were derivatized to trimethylsilyl ethers for quantification by GC-MS using selected-ion monitoring. Both assays were evaluated for consistency of response, precision, accuracy and specificity. Limits of quantification for I and II by HPLC were 15 and 20 ng/ml, respectively; limits of quantification for I and II by GC-MS were 2 and 5 ng/ml, respectively. Both methods were applied to the analysis of clinical samples from oral dosing studies with I.

Pharmacokinetic interactions between arbaprostil and aspirin in humans.

Arbaprostil is an orally active prostaglandin E2 analogue. It has been developed as a drug to treat ulcers induced by non-steroidal anti-inflammatory drugs. In this study, pharmacokinetic interactions between arbaprostil and aspirin were examined in humans after chronic doses of both drugs. Subjects received either arbaprostil (50 micrograms), aspirin (975 mg) or arbaprostil (50 micrograms) and aspirin (975 mg) four times a day for 6 days and one dose on 7th day. Blood and urine samples were collected after the last dose for 6 h. Pharmacokinetic parameters of arbaprostil, aspirin, and salicylate were determined. Coadministration of arbaprostil significantly lowered the area under curve (5.09 +/- 0.32 micrograms hml-1 vs 5.78 +/- 0.29 micrograms hml-1, mean +/- SE, p less than 0.05) and time (0.45 +/- 0.07 h vs 0.70 +/- 0.12 h, p less than 0.05) to reach maximal plasma concentration of aspirin (acetylsalicylate). The pharmacokinetics of salicylate were not changed by arbaprostil, nor were the pharmacokinetics of arbaprostil affected by aspirin. Coadministration of these two drugs did not appear to potentiate the side-effects of either drug. The results suggest that arbaprostil and aspirin may be administered together without clinically significant changes in pharmacokinetics or adverse side-effects.

Irregular retention properties of 21-aminosteroid antioxidants in octylsilane bonded-phase high-performance liquid chromatography.

The retention characteristics of two novel 21-aminosteroid antioxidants, 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9(11)-triene-3,20-dione dimethanesulfonate (I) and 21-[4-(2,5-di-N-diethylamine-2-pyridinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9(11)-triene-3,20-dione hydrochloride (II), in octylsilane bonded-phase high-performance liquid chromatography were examined in detail. Both I and II exhibited irregular retention behaviour which could be explained by the dual retention mechanism model proposed by A. Nahum and Cs. Horváth [J. Chromatogr., 203 (1981) 53]. Unless an amine modifier was added to competitively inhibit association with exposed silanol binding sites, the retention of both compounds in a 70% acetonitrile mobile phase was primarily due to silanophilic interactions. Addition of amine modifiers, lowering the pH, and increasing the sodium ion concentration of the mobile phase all decreased retention times, and modifiers capable of hydrogen bonding diminished peak tailing.

High-performance liquid chromatographic determination of a 21-aminosteroid antioxidant in plasma.

A novel 21-aminosteroid, 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1- piperazinyl]-16 alpha-methylpregna-1,4,9(11)-triene-3,20-dione monomethanesulfonate (I), is under development for the treatment of central nervous system injury in humans. This report describes a reversed-phase high-performance liquid chromatographic (RP-HPLC) method using ultraviolet detection at 254 nm for the determination of I in plasma with low nanogram per milliliter sensitivity. Plasma was deproteinated by mixing with acetonitrile and centrifuging, and I was extracted from the supernatant with disposable bonded-phase columns. The extracts were chromatographed on an octylsilane bonded-phase HPLC column with an acetonitrile-water mobile phase containing triethylammonium acetate, pH5. Quantification was accomplished by peak-height ratio analysis using an analogue of I as the assay internal standard. The method was suitable for the determination of I following a 30 mg/kg intraperitoneal dose in the rat.

Pulmonary extraction and pharmacokinetics of prostaglandin E1 during continuous intravenous infusion in patients with adult respiratory distress syndrome.

Prostaglandin E1 (PGE1) is currently being evaluated in clinical trials to determine its usefulness in the treatment of adult respiratory distress syndrome (ARDS). The drug is administered to ARDS patients by continuous intravenous infusion at dosage rates of up to 30 ng/kg/min for 7 days. The present study was conducted to determine the pulmonary extraction efficiency and pharmacokinetics of PGE1 under these conditions. Plasma levels of PGE1 were determined by high performance liquid chromatography in 14 patients who either had ARDS or were considered to be at risk of developing ARDS following trauma or sepsis. Predose plasma levels of PGE1 were below the detection limit of the assay (50 pg/ml). At a dosage rate of 30 ng/kg/min, pulmonary arterial and systemic arterial plasma levels ranged from 265 to 1,009 pg/ml and 50 to 796 pg/ml, respectively. The pulmonary extraction ratio (Ep) of PGE1 varied from 0.11 to 0.90 and was independent of dose but dependent on cardiac output. The data were adequately described by first-order pharmacokinetic equations which assumed that the lung was the only site of PGE1 clearance. Nine of 10 patients with AaPO2/FlO2 below 510 mm Hg had Ep greater than 0.7 and high pulmonary intrinsic clearance for PGE1 (ca. 250 L/min), but all 4 patients with AaPO2/FlO2 above 510 mm Hg had Ep less than 0.6 and low intrinsic clearance (ca. 37 L/min or less). The intrinsic clearance of the lung for PGE1 in ARDS patients therefore appears to decrease abruptly once a threshold of severe respiratory failure is achieved.

High performance liquid chromatographic determination of thromboxane B2 in human serum as a methoxime-panacylester derivative.

A high performance liquid chromatographic (HPLC) method was developed to measure thromboxane B2 (TxB2) levels in human serum. Serum samples (2 mL) were extracted using solid phase extraction columns in a C 18/silica mode sequencing approach. The internal standard, 6-ketoprostaglandin F1 alpha, was added to the serum extracts. The eicosanoids were doubly derivatized, first with panacyl bromide, then with methoxyamine to form methoxime-panacyl ester derivatives. The eicosanoid derivatives were chromatographed using a reverse phase HPLC system with UV detection (254 nm). Assay linearity was demonstrated with fortified TxB2 standards in 3% bovine serum albumin over a range of 25 to 500 ng/mL (r greater than or equal to 0.994). There was no significant interday difference or bias in assay results for pooled standards at 75, 226 and 376 ng/mL concentrations (p greater than 0.05). Pooled estimates of precision at these levels indicate an assay relative standard deviation 6-9%. The HPLC assay was used to quantitate TxB2 levels in human serum. Results were consistent with previously published values when drug-free serum was analyzed to assess ex vivo TxB2 formation.

Prostaglandin E1 prevents increased lung microvascular permeability during intravascular complement activation in sheep.

Prostaglandin E1 (PGE1) inhibits a variety of functions of activated neutrophils including respiratory burst, release of leukotriene B4, and adherence to endothelial cells. To determine if PGE, alters the pathophysiology of complement-induced lung vascular injury, experiments were conducted in anesthetized sheep with lung lymph fistulas given a 1-hour infusion of zymosan-activated plasma. PGE1 (30 ng/min/kg) or its saline vehicle was infused intravenously for 90 minutes beginning 30 minutes before the infusion of activated plasma. PGE1 had no effect on leukocyte count, the initial hypoxemia and thromboxane A2 release, or the development of acute pulmonary hypertension. However, PGE1 prevented steady-state increases in lung lymph flow that in vehicle-treated sheep signaled an increase in lung microvascular permeability. Furthermore, extraction of PGE1 by pulmonary endothelial cells was unaffected by the infusion of activated plasma. We propose that PGE1 prevented the increase in lung vascular permeability by inhibiting adherence of activated neutrophils to endothelial cells.

Plasma levels of the prodrug, arbaprostil, [(15R)-15-methylprostaglandin E2], and its active, antiulcer (15S) epimer in humans after single dose oral administration.

Arbaprostil [(15R)-15-methylprostaglandin E2] is an antiulcer prodrug being evaluated for the treatment of gastric and duodenal ulcers in humans. It epimerizes in acidic gastric fluid to produce the biologically active form, (15S)-15-methyl-PGE2, which acts directly on the gastric mucosa and possesses both gastric acid antisecretory and cytoprotective properties. Because of its local mode of action, plasma levels of the two epimers may have greater relevance to drug safety than to therapeutic efficacy. In the present study, plasma concentrations of both 15-methyl-PGE2 epimers resulting from a gastric acid antisecretory dose of arbaprostil oral solution (50 micrograms) were measured in eight male volunteers having sufficient gastric acidity for prodrug activation (pH less than 3). Arbaprostil was determined with a newly developed RIA having a sensitivity of 10 pg X mL-1. The accuracy of the RIA was confirmed by parallel analysis of plasma samples by HPLC. (15S)-15-Methyl-PGE2 was also determined by HPLC. Arbaprostil was both rapidly absorbed and eliminated (tmax of 15-30 min and plasma t1/2 of 20 min), but there was large intersubject variability in its observed maximum plasma concentration (38 to 348 pg X mL-1). The concentration of (15S)-15-methyl-PGE2 did not exceed 25 pg X mL-1 In six subjects and 50 pg X mL-1 in the remaining two subjects. The significance of these results is discussed.

Determination of prostaglandin E1 in plasma with picogram per millilitre sensitivity by double antibody extraction and column switching high-performance liquid chromatography.

Prostaglandin E(1) (PGE(1)) is a vaso- and arteriodilator that is used for the experimental treatment of a variety of vascular diseases. A high-performance liquid chromatography (HPLC) method was developed for the purpose of quantifying PGE(1) in the plasma of patients undergoing constant infusion therapy. Plasma (0.5 ml) was extracted with a double antibody precipitation technique and the PGE(1)-immunoprecipitate was isolated and washed by repeated resuspension and centrifugation. PGE(1) was recovered from the precipitate by extraction with acetonitrile and derivatized with panacyl bromide for determination on a heteromodel column switching HPLC system with fluorescence detection. The content of PGE(1) in immunoextracts was determined by HPLC peak height comparison against a standard curve of PGE(1) peak height vs amount derivatized. The result was corrected for the plasma extraction efficiency (determined with radiolabelled PGE(1)) to obtain the plasma concentration. Standard curves were linear from 25 to 400 pg (r > 0.99) and the y-intercepts were not significantly different from zero (p < 0.05). The immunoextraction recovery from six human donors was 63.5 +/- 2.0% (S.D., n = 18). The quantification limit of the method was 50 pg ml(-1) (signal-to-noise ratio 3:1), at which the estimated assay relative standard deviation was 18%. The utility of the method for the measurement of PGE(1) plasma levels during constant intravenous infusion was demonstrated in a dog study.

Determination of (15R)- and (15S)-15-methylprostaglandin E2 in human plasma with picogram per milliliter sensitivity by column-switching high-performance liquid chromatography.

(15R)-15-Methylprostaglandin E2 (PGE2) is a pro-drug under evaluation for the treatment of acute upper gastrointestinal hemorrhage and gastrointestinal cytoprotection. It is converted in acid (e.g., gastric fluid) to its active 15S epimer. Both epimers are found in human plasma at low pg/ml levels following oral dosing. A high-performance liquid chromatographic (HPLC) method was developed for the simultaneous analysis of (15R)- and (15S)-15-methyl-PGE2 in human plasma. The method combined off-line solid-phase extraction and reversed-phase HPLC clean-up with panacyl bromide derivatization and subsequent analysis using a heteromodal column-switching technique. Assay linearity was demonstrated over a range of 10-200 pg/ml for both 15-methyl-PGE2 epimers (r greater than or equal to 0.995). There were no significant inter-day differences in assay results for either epimer at 50 and 25 pg/ml (p greater than 0.05), with pooled estimates of precision at these levels producing relative standard deviations of less than or equal to 8% and less than or equal to 12%, respectively. The method quantitation limit (signal-to-noise ratio 5:1) for both epimers was 10 pg/ml when processing 3 ml of plasma. The analysis procedure was shown to be useful for quantifying at or below 10% of the (15R)-15-methyl-PGE2 maximum plasma concentration following a 50-micrograms oral dose in three human volunteers. For the same three subjects, however, the plasma concentration of (15S)-15-methyl-PGE2 did not exceed the quantitation limit of 10 pg/ml.

Isomodal column switching high-performance liquid chromatographic technique for the analysis of ciglitazone and its metabolites in human serum.

The application of isomodal column switching high-performance liquid chromatography as an alternative to gradient elution was investigated for the analysis of ciglitazone , a potential oral antidiabetic agent, and its monohydroxyl metabolites in human serum. A high-performance liquid chromatographic apparatus was designed to perform on-line fractionation of the serum extract into non-polar (drug) and polar (metabolite) fractions which were then automatically routed into individually optimized, isocratic, reversed-phase high-performance liquid chromatographic systems for simultaneous analysis. Sample fractionation was performed with a reversed-phase guard column, and solvent routing was accomplished with microprocessor-controlled switching valves. Serum was extracted for analysis by a one-step mode sequencing procedure using disposable bonded-phase columns, and quantitation was accomplished with spiked serum standards. Performance specifications of the method were defined for precision, accuracy, linearity, and sensitivity. The column switching method was found to be both expedient and reliable, and it may have general utility for the routine, quantitative analysis of drug/metabolite mixtures that cannot be assayed by simple isocratic elution methods.