Isocratic Reversed-phase High Performance Research Articles

Determination of Fluvoxamine and Paroxetine in Human Serum with High-Performance Liquid Chromatography and Ultraviolet Detection

We optimized an isocratic reversed phase high performance liquid chromatography with ultraviolet detection to quantify, simultaneously, the selective serotonin reuptake inhibitors (SSRIs) fluvoxamine and paroxetine in human serum. Fluvoxamine was used as the internal standard for the determination of paroxetine, and paroxetine served as internal standard for the fluvoxamine assay. The method involved a pre-column technique for the on-line liquid-solid extraction with direct injection of serum samples and for their pre-concentration. Automation was achieved by column switching. The chromatographic separation was performed on an Ultrasep ES 100 CN-column with acetonitrile/methanol/phosphate buffer (58/19/23, v/v/v) as mobile phase. A linear relationship (r2 > 0.99) was noted between the concentrations of fluvoxamine or paroxetine and the detector signal. The lower limits of detection in human serum of fluvoxamine and paroxetine were 5 and 2 μg/L, respectively. The accuracy of the quality control samples deviated by m± 8% with a within-day and between-day precision of less than 12.1%, and 10.5% for fluvoxamine and paroxetine, respectively. The method is presently being applied in our clinic for the routine therapeutic drug monitoring of both SSRIs.

Isocratic mixed mode liquid chromatographic separation of phospholipids with octadecylsilane-silica stationary phases

Molecular species of phosphatidylcholine, phosphatidylethanolamine and phosphatadic acid were resolved by isocratic reversed phase high performance liquid chromatography (HPLC) using mobile phases of methanol—isopropanol containing para-toluenesulfonic acid ( p-tsa). Separation by both non-polar fatty acid chain length and by polar head group functionality was achieved concurrently upon a commercially available octadecylsilane (C18) column endcapped with trimethylsilane (C1) groups. Using a mobile phase of 97.5:2.5 methanol:isopropanol with 7Om M para-toluenesulfonic acid ( p-tsa) at a pH of approximately 1, twelve phospholipid species comprised of four tail group classes (dilauroyl-,dimyristoyl-, dipamitoyl- and distearoyl-) and three head group speciations (phosphatidylcholine, phosphatidylethanolamine and phosphatadic acid) were separated. The column was then exposed to the acidic mobile phase for 48 hours continuously during which the bound phase underwent severe acid-induced hydrolysis, after which the separation of the twelve analytes resulted in the separation of the phospholipid species by non-polar tail group alone. The experimental results are discussed in terms of potential separation mechanisms including dependency of the separation on adsorption of the counter ion into the stationary phase, residual acidic silanol group interactions, and potential interactions of the surface active phospholipids with C1 groups.

Increased concentrations of endogenous 13- cis- and all- trans-retinoic acids in diffuse idiopathic skeletal hyperostosis, as demonstrated by HPLC

Endogenous 13- cis- and all- trans-retinoic acids have been quantitated in human serum using a solvent extraction procedure followed by isocratic reversed phase high performance liquid chromatography and UV detection. In healthy adults, after an overnight fasting period, the concentrations of 13- cis- and all- trans-retinoic acids yielded 5.3 ± 2.43 nmol/l and 11.8 ± 3.3 nmol/l, respectively (mean ±SD). The method has been successfully applied to the analysis of both isomers in serum from patients with idiopathic skeletal hyperostosis in whom, the 13- cis- as well as all- trans-retinoic acid levels were raised as compared to the control group.

Isocratic reversed phase high performance liquid chromatography determination of twelve natural corticosteroids in serum with on‐line ultraviolet and fluorescence detection

An isocratic reversed phase high performance liquid chromatography procedure utilizing ultraviolet and fluorescence detectors linked in series is described for the analysis of cortisone (E), cortisol (F), corticosterone (B), 11-deoxycortisol (S), 11-deoxycorticosterone (DOC), androstenedione (A), testosterone (T), 17-hydroxyprogesterone (17-OHP), progesterone (P), estriol, estradiol, estrone, prednisone acetate and dexamethasone acetate in serum. Serum specimens were extracted with ethyl ether. The optimized mobile phase was methanol + tetrahydrofuran + water (26:18:56, v/v/v). A Shim-pack ODS column was used. The recoveries were 80 to 103%. Intra- and inter-day coefficient of variance were less than 8%. The detection limit is 0.5 pmol per injection volume for estriol, estradiol, E, F and B; 1 pmol for S, A, DOC and estrone; 2 pmol for T and 17-OHP; and 4 pmol for P. Serum from normal subjects and patients with congenital adrenal hyperplasia due to 21- or 17-hydroxylase deficiency were measured, as well as samples of maternal and umbilical cord serum.

Quinidine oxidative metabolism. Identification and biosynthesis of quinidine 10,11‐dihydrodiol stereoisomers

The isocratic reversed phase high performance liquid chromatographic method proposed for quinidine metabolic studies facilitates particularly the separation of 10(R) and (S) isomers of quinidine 10,11-dihydrodiols. The finding of each of these forms following a new synthetic pathway allows us to identify and quantify them in biological fluids. These two isomers have especially been observed in rat bile and hepatocyte secretions. The metabolic inducing effect of phenobarbital on the oxidative metabolism of quinidine is verified in rat isolated hepatocytes. Simultaneous secretion of the two dihydrodiols is also verified in human urine by a gas chromatography/mass spectrometry procedure.

Optimization of an Isocratic Reversed Phase Liquid Chromatographic System for the Separation of Fourteen Steroids Using Factorial Design and Computer Simulation

An optimization strategy for an isocratic reversed phase high performance liquid chromatographic system (RP-HPLC) is described. Factorial design and a computer program are used to predict the retention time and resolution of fourteen steroids. An optimized rapid (less than 25 min) isocratic RP-HPLC system for the satisfactory separation of cortisone, cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17 alpha-hydroxyprogesterone, progesterone, androstenedione, testosterone, estrone, estradiol, estriol, prednisone acetate and dexamethasone acetate has been developed using this strategy through eight experiments.

Revrsed Phase High Performance Liquid Chromatographic Analysis of Triphenylphosphine in a Reaction Mixture

Abstract An isocratic reversed phase high performance liquid chromatographic method is described for monitoring concentrations of triphenylphosphine (TPP) and other compounds involved in an oxygen-transfer reaction catalyzed by a molybdenum complex. Using an inexpensive octadecylsilane column and a mobile phase consisting of aqueous acetonitrile, good separations and linear chromatographic responses were achieved for solutions of TPP, pyridine and each of their oxides in the mobile phase with concentrations in the range of 0.0012 – 0.12 grams/100 mL. No evidence for TPP precipitation was observed during the analysis of supersaturated solutions containing TPP at concentrations as high as 0.27 grams/100 mL. Chromatograms of more concentrated TPP solutions exhibited a second peak corresponding in retention time to TPP-oxide. Contrary to the interpretation of a similar effect in a previous study, evidence is presented indicating that this second peak is not the result of decomposition of TPP to its oxide, but...

Measurement of Angiotensin II Concentration in Rat Plasma: Pathophysiological Applications

A very reliable isocratic reverse phase high performance liquid chromatography (HPLC) system has been developed to separate angiotensins, which combined with a very sensitive radioimmunoassay, provided precise measurements of the endogenous angiotensin II (AII) concentration in the rat plasma in different experimental circumstances. The overall recoveries of AII were 95.2 +/- 15.8% (means +/- SD) when 10 pg of this peptide was added to 1 ml of human plasma. The coefficient of variation for within-assay precision was 10% (n = 6). The plasma AII, measured by this method, of normal male pentobarbital-anesthetized rats was 53.0-141.6 pg/ml (mean: 103.9 +/- 29.7 pg/ml). The plasma AII of rats fed a sodium deficient diet was 300.0 +/- 100.6 pg/ml, while that of rats given oral Enalapril, an angiotensin converting enzyme (ACE) inhibitor, for 1 week was 35.7 +/- 28.0 pg/ml. The plasma AII of bilaterally nephrectomized rats was 2.7 +/- 2.9 pg/ml 24 hours after nephrectomy and below the detection limit 48 hr after nephrectomy. This method, therefore, can be used to study AII in different pathophysiological states or after treatment with renin-angiotensin system inhibitors.

A highly specific detection method for diethylstilbestrol in bovine urine by radioimmunoassay following high performance liquid chromatography.

A specific radioimmunoassay (RIA) for diethylstilbestrol (DES) is presented following purification of sample extracts by isocratic reversed phase high performance liquid chromatography. Applications are given of a forensic investigation for the control of DES in bovine urine. Specificity of HPLC-RIA is compared with that of RIA's with other (chromatographic) purification procedures and with gas chromatography-mass spectrometry. Implications of the use of these techniques in practice and the use of various DES-antisera are discussed.

Lack of metabolism of timolol by ocular tissues.

The ocular metabolism of timolol has been studied in the iris/ciliary body, vitreous humor, neuro-retina and cornea from an albino rabbit, pigmented rabbit and cynomolgus monkey by incubating tissues isolated from these animals with radiolabeled drug for 20 hours at 37 degrees C in Hanks' balanced salts solution. After removal of the tissues by centrifugation and subsequent deproteinization, supernatants were mixed with unlabeled timolol and analyzed by isocratic reverse phase high performance liquid chromatography with liquid scintillation counting and ultraviolet detection. Although this analytical system readily demonstrated the production of timolol metabolites by rat liver, the production of timolol metabolites by ocular tissues could not be demonstrated. With most samples high recoveries of unmetabolized timolol were obtained. The lowest level of recovery of unmetabolized drug as compared to a control (drug added after the tissue had been incubated) was obtained for the iris/ciliary body from the pigmented rabbit suggesting that pigment had bound the drug. Our results suggest that deleterious side effects sometimes seen with the drug or its lack of efficacy with rabbits is not due to ocular metabolism by these tissues.

Determination of 4,4'-alkyl- and alkoxy-disubstituted azoxybenzenes by isocratic reverse phase high performance liquid chromatography

Determination of 4,4'-alkyl- and alkoxy-disubstituted azoxybenzenes by isocratic reverse phase high performance liquid chromatography

Separation of five major alkaloids in gum opium and quantitation of morphine, codeine, and thebaine by isocratic reverse phase high performance liquid chromatography.

Separation of five major alkaloids in gum opium and quantitation of morphine, codeine, and thebaine by isocratic reverse phase high performance liquid chromatography.