Chromatographic Estimation Research Articles

Chromatographic Estimation of Drug Disposition Properties by Means of Immobilized Artificial Membranes (IAM) and C18 Columns

Chromatographic retention measurement in immobilized artificial membranes (IAMs) is considered a fast and reliable method to predict biological properties (drug distribution) because of the IAM structure, which consists of phospholipid analogues bonded covalently to silica particles. A new parameter (d) is proposed to estimate the similarity between IAM columns, conventional HPLC columns, and drug distribution systems, and thus the performance of chromatographic systems to predict drug distribution. An IAM.PC.DD2 column has been used for this study, together with two XTerra columns (MSC18 and RP18), at several acetonitrile-water mobile phases. According to the d parameter, good correlations should be obtained between chromatographic systems (both IAM and C18) and octanol-water partition coefficient (log P), and thus both types of columns could be used to obtain log P values. The IAM.PC.DD2 system shows a close similarity to human skin partition, tadpole narcosis, and blood-brain permeability processes, showing that it can be useful as a model for these biological processes. Controversially, it is shown that human skin permeation is more similar to C18 partition than to IAM partition. Other biological processes such as blood-brain distribution and tissue-blood partition show a poor similarity to IAM and C18 systems, demonstrating that estimation of these drug distribution processes by chromatographic measurements may not be adequate.

Analysis of Free Hemoglobin Level and Hemoglobin Peptides from Human Puerperal Uterine Secretions

Hemocidins are a novel class of antibacterial peptides generated proteolytically from hemoglobin. These peptides play a particularly important role in maintaining vaginal homeostasis during menstrual bleeding. To investigate the hemoglobin fragmentation process during the last stages of pregnancy, we examined uterine secretion (lochia) samples from a group of 22 healthy women who underwent cesarean delivery at term. Patients were divided into three groups: (1) the elective cesarean deliveries without symptoms of spontaneous labor, (2) the nonelective cesarean deliveries with spontaneous beginning of labor, and (3) the nonelective cesarean deliveries during advanced labor. The samples were subjected to chromatographic estimation of free hemoglobin and peptides. In three representative patients the identification of all lochial peptides was performed. All samples contained a significant amount of free hemoglobin and its level increased with labor progression. The presence of peptide fractions was also detected in most lochia samples. They were confirmed to be human hemoglobin fragments, almost identical to the recently described bactericidal hemocidins from menstrual discharge. The level of peptides also increased during labor. The subgroup with advanced labor demonstrated the highest amount of hemocidins. The presented results prove that proteolysis of free hemoglobin in the female upper reproductive tract begins together with the clinical symptoms of normal labor. We speculate that cesarean delivery affects molecular mechanisms involved in antibacterial hemocidins generation and, in effect, might be responsible for the increased risk of gynecologic infections in cesarean deliveries.

Chromatographic estimation of the soil-sorption coefficients of organic compounds

Soil-sorption coefficient ( K OC) assessment is essential before placing a new chemical substance on the market. Since experimental K OC measurements are tedious, time-consuming and costly, alternative approaches to estimating K OC from other descriptors have been proposed. We review the use of chromatographic retention (log k) values, obtained in different types of chromatographic systems, as descriptors to establish predictive log K OC-log k models. We highlighted critical aspects of such models and performed a comparative study using data in the literature. We compare two strategies to deal with the large variability of experimental log K OC data. We contrast the results of this study with the main conclusions reported in the literature.

Noise-Stress-Induced Brain Neurotransmitter Changes and the Effect of Ocimum sanctum (Linn) Treatment in Albino Rats

In this modern world, stress and pollution are unavoidable phenomena affecting the body system at various levels. A large number of people are exposed to potentially hazardous noise levels in daily modern life, such as noise from work environments, urban traffic, and household appliances. A variety of studies have suggested an association between noise exposure and the occurrence of disorders involving extra-auditory organs such as disorders of the nervous, endocrine, and cardiovascular systems. In this study, Wistar strain albino rats were subjected to 100 dB broadband white noise, 4 h daily for 15 days. The high-pressure liquid chromatographic estimation of norepinephrine, epinephrine, dopamine, and serotonin in discrete regions of the rat brain indicates that noise stress can alter the brain biogenic amines after 15 days of stress exposure. Ocimum sanctum (OS), a medicinal herb that is widely claimed to posses antistressor activity and used extensively in the Indian system of medicine for a variety of disorders, was chosen for this study. Administration of the 70% ethanolic extract of OS had a normalizing action on discrete regions of brain and controlled the alteration in neurotransmitter levels due to noise stress, emphasizing the antistressor potential of this plant.

Chromatographic estimation of filler surface energies and correlation with photodegradation of kaolin filled polyethylene

Inverse gas chromatography (IGC) has been used to investigate the surface properties of two calcined kaolins and of polyethylene composites containing calcined kaolin. The results reveal differences in the two kaolins that were not found in XPS spectra nor in their influence on the melting behaviour of the polymer. The kaolin that had a markedly higher surface energy and stronger Lewis acid behaviour was found to shower greater photooxidative degradation in accelerated weathering tests. This novel application of IGC derived surface characterisation of solids further extends the usefulness of the method in this type of work.

A possible role of poly-3-hydroxybutyric acid in antibiotic production in Streptomyces.

The occurrence of poly-3-hydroxybutyric acid (PHB) in 12 different strains of the genus Streptomyces was investigated. Gas chromatographic estimation indicated that all the strains produced PHB and the range of maximum PHB accumulation was between 1.5 and 11.8% dry cell weight. PHB was isolated from Streptomyces coelicolor A3(2) M145 and characterized using Fourier transform-infrared (FT-IR) spectroscopy. The correlation between PHB utilization and antibiotic production in S. coelicolor A3(2) M145, was studied; results indicated a possible role of PHB as a carbon reserve material used for antibiotic production.

Development and validation of a new high-performance liquid chromatographic estimation method of meloxicam in biological samples

A simple, HPLC method was developed to estimate meloxicam (COX-2 inhibitor) using piroxicam as the internal standard. The mobile phase containing methanol, acetonitrile and an aqueous solution of diammonium hydrogenorthophosphate (50 m M) in the ratio of 4:1:5 was pumped at the rate 1 ml/min. Lichrocart RP-18 (125×4 mm) was used as an analytical column and the analytes were detected at 364 nm using a UV detector. Acidified plasma samples were extracted with chloroform, evaporated to dryness, reconstituted in the mobile phase and then a volume of 10 μl of the prepared sample was injected in the column. The retention time of meloxicam and piroxicam was found to be 2.7 and 1.9, respectively. This method showed an accuracy of 102.3% at 0.52 μg/ml and was capable of detecting a minimum concentration of 0.029 μg/ml meloxicam from biological samples. The analytical method was successfully utilized for estimating meloxicam in biological samples.

Isocratic, simultaneous reversed-phase high-performance liquid chromatographic estimation of six drugs for combined hypertension therapy

We report an isocratic, HPLC procedure for assay of the orally administered hypertension drugs [atenolol, amlodipine, nifedipine, nitrendipine, nimodipine and felodipine given in retention order] of which atenolol, an aryloxypropanolamine β-blocker is administered with anyone of the other five dihydropyridine calcium channel blockers in combined hypertension therapy. The drugs were dissolved in methanol and 20 μl of a mix of the drugs was injected onto a reverse phase JASCO-metaphase ODS (250×4.0 mm) 5 μ column. Any one of the six drugs could be used as the internal standard. The drugs were resolved by elution with a pH 4.5 equivolume mobile phase of acetonitrile–0.01 M KH 2PO 4, with pH adjustments done with H 3PO 4 (flow-rate 1.5 ml min −1). The column effluent was monitored at 250 nm. The detector response (peak height ratio) was linear in the dynamic range of 25–3200 ng ml −1 of these drugs, with the detection limits at ∼15 ng ml −1. Full statistical evaluation of the data including linear regression (least-square fit) analysis was performed. The suggested procedure has the advantage that all the five dihydropyridine derivatives can be quantified alone or in formulation with atenolol.

Chromatographic quantitation of the hydrophobicity of ionic compounds by the use of micellar mobile phases

Many biologically active compounds of interest in structure–activity relationships are ionic at physiological pH. However, ionic organic compounds are only weakly or not retained in conventional RPLC which impedes the chromatographic estimation of their hydrophobicity and the development of quantitative retention–activity relationship studies. The use of micellar mobile phases allows the retention of ionic compounds. Hydrophobic and electrostatic forces govern the retention of ionic compounds in micellar liquid chromatography. In this paper three different retention models log k–log P for ionic compounds are tested ( P=partition coefficient). The retention model (log k= a log P+ b α+ c) which includes the hydrophobicity and the molar total charge of compound at a given pH value has proven to be valid for all types of compounds tested, catecholamines, local anesthetics, diuretics and o-phthalaldehyde–N-acetyl- l-cysteine amino acid derivatives.

Chromatographic estimation of iothalamate and p-aminohippuric acid to measure glomerular filtration rate and effective renal plasma flow in humans

Iothalamate (IOT) clearance and p-aminohippuric acid (PAH) clearance are used for estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid method is described for simultaneous determination of IOT and PAH in the same chromatogram in the serum and urine of humans. The mobile phase consisted of methanol–50 m M sodium monobasic phosphate with 0.5 m M tetrabutyl ammonium hydrogen sulfate (18:82, v/v), pumped at a rate of 0.8 ml/min on a C 1 8 reversed-phase column. Samples of serum and urine were deproteinized with two volumes of acetonitrile containing the internal standard, p-aminobenzoic acid (PABA). The UV detector was set at 254 nm and peak height ratios of PAH or IOT to PABA were calculated with an integrator. Precision and accuracy were within 15% for both PAH and IOT. The recovery of PAH in urine and serum were 94% and 91%, respectively. For IOT the corresponding recoveries were 93% and 92%, respectively. This method clearly distinguishes acetyl-PAH from PAH and has been validated in healthy volunteers.

The kinetic behaviour of [ 3H]DOPA in living rat brain investigated by compartmental modelling of static autoradiograms

The kinetic behaviour of [ 3H]DOPA in living rat brain was investigated by compartmental modelling of measured activities from combined metabolite pools in a time-series (180 min) of static autoradiograms from right cerebral hemispheres. Two models of [ 3H]DOPA uptake and metabolism that incorporated the removal of the decarboxylation product, [ 3H]dopamine, from brain were significantly more accurate than a model in which [ 3H]dopamine accumulated irreversibly in situ. Present estimates of [ 3H]DOPA kinetic constants were compared to previously published results based on the analysis of measured activities from individual metabolite pools separated by chromatographic fractionation of [ 3H]DOPA metabolites in the left cerebral hemispheres of the same rats. Autoradiographic estimates of DOPA decarboxylase activity with respect to [ 3H]DOPA in brain ( k 3 DOPA) were under-estimated several-fold relative to chromatographic estimates; this discrepancy is explained by post-mortem enzyme activity and omission of biological compartments from the models. However, autoradiographic estimates of the unidirectional blood–brain clearance of [ 3H]DOPA ( K 1 DOPA) and monoamine oxidase activity with respect to [ 3H]dopamine in brain ( k 7 DA′) agreed with chromatographic estimates. This concordance represents the first empirical validation of compartmental modelling of autoradiographic data as a method for quantitatively investigating the kinetic behaviour of radiolabelled l-DOPA in living mammalian brain.

Comparative study of release kinetics of pheromone from polymer dispensers

Polymer membranes loaded with agrochemicals are used as controlled release monolithic dispensers in pest control. Plasticized poly(vinyl chloride) dispensers loaded with a fixed concentration of gossyplure pheromone were prepared by a solution-casting method. A solvent trap system was designed and fabricated to collect the gossyplure released from the dispenser at desired intervals of time. The release of pheromone was estimated quantitatively by using high-performance liquid chromatography. The rate of release is found to be slightly higher in the first 12 h and then it became steady, obeying Fick's law of diffusion. The diffusion coefficient of the pheromone is observed to be directly proportional to the concentration of the plasticizer in the dispenser. Simultaneously, the rate of release of pheromone from the dispenser was also determined by a gravimetric desorption method. The diffusion coefficient data obtained from the gravimetric desorption method was found to be fairly comparable to that obtained from high-performance liquid chromatographic estimation. The results indicate the authenticity and reliability of the gravimetric desorption method. The dispensers were found to release gossyplure at reasonably controlled rates even after 30 days. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 64: 1373–1380, 1997

Comparative study of release kinetics of pheromone from polymer dispensers

Polymer membranes loaded with agrochemicals are used as controlled release monolithic dispensers in pest control. Plasticized poly(vinyl chloride) dispensers loaded with a fixed concentration of gossyplure pheromone were prepared by a solution-casting method. A solvent trap system was designed and fabricated to collect the gossyplure released from the dispenser at desired intervals of time. The release of pheromone was estimated quantitatively by using high-performance liquid chromatography. The rate of release is found to be slightly higher in the first 12 h and then it became steady, obeying Fick's law of diffusion. The diffusion coefficient of the pheromone is observed to be directly proportional to the concentration of the plasticizer in the dispenser. Simultaneously, the rate of release of pheromone from the dispenser was also determined by a gravimetric desorption method. The diffusion coefficient data obtained from the gravimetric desorption method was found to be fairly comparable to that obtained from high-performance liquid chromatographic estimation. The results indicate the authenticity and reliability of the gravimetric desorption method. The dispensers were found to release gossyplure at reasonably controlled rates even after 30 days. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 64: 1373–1380, 1997

Acid-catalyzed cyclocondensation of nitriles. Part IV. Synthesis and spasmolytic activity of 1-substituted 3-aminoisoquinolines and their derivatives

Biologically active compounds with an isoquinoline nucleus are widely used in medicine (papaverine, No-Spa, salsoline, etc.). Earlier, we have developed a simple and efficient method for the synthesis of 3-aminoisoquinolines and the corresponding 3-iminocarbonylamino derivatives [ I, 2]. The purpose of this work was to obtain acyl derivatives of 3-aminoisoquinolines, substituted at the amino group, and study the spasmolytic activity of both 3-aminoisoquinolines and their derivatives. The initial 3-aminoisoquinolines (IIIa-IIIc) were synthesized using a method described previously [ 1, 2]. The first step of this procedure consists in the acid-catalyzed cyclocondensation of arylacetonitriles. This is a three-stage process (see Scheme 1) involving the formation of intermediate Nimidoylnitrilium ions (II) and 3-aminoisoquinoline (III); the reaction mechanism was considered in detail elsewhere [1 ]. Despite a rather hitch yield of final 3-aminoisoquinolinyl aminoimides IV (50 - 72%), the products always contain unreacted initial nitriles and 3-aminoisoquinolines (dimeric products) [1, 2]. Below we will demonstrate that increasing the duration of the first stage leads to more complete consumption of the reagents. Chromatographic estimates show that the content of IIIa in the reaction mass eventually decreases to 3 - 5%, while the yield of amidine IVa, with an allowance for the conversion of 3,4-dimethoxyphenylacetonitrile, amounts to 87%. The content of 3-aminoisoquinolines in the reaction mixture varies depending on the conditions, reaching a maximum by the end of the low-temperature reaction stage [3]. However, taking into account difficulties encountered in the isolation of these compounds, it is expedient to terminate reaction upon the stage of 3-aminoimidoylisoquinolines IV, with their subsequent conversion into corn

Liquid Chromatographic Estimation of Octanol/Water Partition Coefficients with a High Efficiency, Nonporous, Ultrasmall Particle Size Reverse Phase Stationary Phase

Abstract HPLC retention data, obtained over a range of organic modifier fractions φ in binary mobile phases, can be used to estimate a compounds octanol/water partition coefficient (log Po/w). Such a determination is facilitated by a high efficiency, low capacity column. The utility of a nonporous, ultrasmall particles size (1.5 μ), C18 stationary phase capable of such performance was assessed using aqueous mobile phases containing either acetonitrile or methanol. In general, compounds were effectively eluted from this stationary phase at lower values of φ than with more typical stationary phases, thus increasing the accuracy and speed with which log Kw, the capacity factor in a 100% aqueous mobile phase, could be determined. Values of log Kw obtained for seventeen model compounds were highly correlated with the compound's log P0/w. Comparison of retention trends in the methanol and acetonitrile mobile phases suggests differences in the retention mechanisms for both organic modifiers.

On the precise estimation of R M values in reversed-phase thin-layer chromatography including aspects of pH dependence

In order to improve the application of reversed-phase thin-layer chromatography (RP-TLC) for the chromatographic estimation of drug lipophilicity, some aspects of measuring true R M values [log (1/ R F - 1)] are considered in the present investigation: (1) An optimization of experimental conditions, including the importance of temperature and humidity, as well as densitometric evaluation of spots is presented. (2) The estimation of thermodynamically true R M values is described; it is shown that in case of high modifier contents preloading effects induce pronounced deviations of R F from R F ′ values. Only the latter allow the calculation of true R M values. (3) The influence of solvent pH on R M values is negligible for pure partition chromatography in the case of low modifier contents; with increasing modifier contents polar adsorption becomes more prominent; under these conditions an influence of pH on R M in the case of strong bases is detected.

Gas chromatographic profiles of plasma total lipids as indicators of dietary history: Correlation with carbohydrate and alcohol intake based on 24-h dietary recall

Quantitative gas chromatographic estimates of the major lipid classes and molecular species in fasting plasma were correlated with total carbohydrate, starch, fibre, sucrose and alcohol intake based on 24-h dietary recall. Spearman coefficients ( r s) and tests of significance ( P) were obtained for groups of 775 males and 471 females aged 20–59 years from a Toronto-McMaster Lipid Research Clinics Population Study. The most significant correlations varying from r s 0.1 to 0.2 and P 0.001 to 0.0005 ( n = 400–773) were between increased intake of alcohol and increased ratios of C 50 C 54 triacylglycerols, C 34 C 36 phosphatidylcholines and phosphatidylcholine/free cholesterol ( PC FC ) of plasma. Increase in total dietary carbohydrate, starch and fibre correlated with decreasing C 50 C 54 triacylglycerol, C 34 C 36 phosphatidylcholine and PC FC ratios ( r s = −0.1–0.2; P < 0.002–0.04; n = 400–773). In contrast, consumption of high levels of alcohol was associated with increasing C 50 C 54 triacylglycerol, C 34 C 36 phosphatidylcholine and PC FC ratios. A high intake of alcohol (50–150 ml per day) distinguished itself from other simple carbohydrate-induced lipid profiles by its marked effect on increased C 50 C 52 triacylglycerol and PC FC ratio.

Plasma Gaba–Like Activity in Rats With Hepatic Encephalopathy Is Due to Gaba and Taurine

Significant discrepancy exists between radioreceptor and high-performance liquid chromatographic estimates of plasma GABA concentrations in animal models of hepatic encephalopathy. A possible explanation for this discrepancy is the presence in plasma of a substance that can inhibit [3H]-GABA binding but is not GABA itself. The aim of this study was to determine whether any of the amino acids that are increased in the plasma of animal models of acute and chronic hepatic encephalopathy (glutamine, glutamate, phenylalanine, tyrosine, citrulline and taurine) can significantly inhibit [3H]-GABA binding and contribute to the GABA-like activity of plasma aliquots. At final assay concentrations equivalent to plasma concentrations found in hepatic encephalopathy, only taurine was found to significantly inhibit [3H]-GABA binding to whole rat brain synaptosomal membranes (36% inhibition at 30 mumol/L final assay concentration). The concentration of taurine that resulted in 50% inhibition (final assay concentration) was 158 mumol/L, corresponding to a plasma concentration of 1.58 mmol/L. Taurine inhibition of [3H]-GABA binding was competitive because the receptor density was unaltered, but the receptor affinity decreased with increasing concentration of taurine. Plasma GABA-like activity (determined by radioreceptor assay) and plasma GABA and taurine concentrations (determined by high-performance liquid chromatography) were measured in 18 rats with acute or chronic hepatic encephalopathy and 9 control rats. Plasma GABA-like activity and plasma GABA and taurine concentrations were significantly increased in rats with hepatic encephalopathy compared with control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Participation of electrolyte cations in albumin adsorption onto negatively charged polymer latices

The participation of electrolyte cations in the adsorption of bovine serum albumin (BSA) onto polymer latices was investigated. The latices used were hydrophobic polystyrene (PS), and hydrophilic copolymers, i.e., styrene (St)/2-hydroxyethyl methacrylate(HEMA) copolymer [P(St/HEMA)] and styrene/acrylamide (AAm) copolymer [P(St/AAm)]. Three kinds of electrolyte cations (Na+, Ca2+, Mg2+) were used as the chloride. The amount of BSA adsorbed in every cation medium showed a maximum near the isoelectric point (iep, pH about 5) of the protein. The amounts of BSA adsorbed onto copolymer latices (except in the acidic pH region lower than the iep) were considerably smaller than that onto PS latex because of the steric repulsion and the decrease in the hydrophobic interaction between BSA and copolymer latices. In the acidic pH region, there was little difference in the amount of BSA adsorbed in every cation medium. However, in the pH region higher than the iep, the amounts of BSA adsorbed (particularly onto PS latex) in divalent cations (Ca2+ and Mg2+) media were relatively greater compared with that in a monovalent (Na+) one. This result was interpreted on the basis of the differences in such effects of electrolyte cations as dehydration power, suppression of the electrostatic repulsion, and binding affinity to BSA molecule. Ion Chromatographic estimation of the amounts of electrolyte cations captured upon BSA adsorption (in pH > 5) revealed that divalent cations were incorporated into the contact interface between the latex and BSA molecule so as to prevent the accumulation of anion charge and facilitate the protein adsorption.

High-performance liquid chromatographic estimation of eluates from denture base polymers

A quantitative analytical method using high-performance liquid chromatography is developed to estimate elution profiles of dental acrylic polymers used for denture bases. The method is applied to elution studies in distilled water of resins polymerized by different representative methods. Elution properties or ‘leachabilities’ vary depending on polymerization conditions. Methyl methacrylate, methacrylic acid and benzoic acid are eluted from autopolymerized resins at the highest eluate concentrations and are followed by heat- and microwave-polymerized resins. These results appear to be comparable to residual amounts of the eluates in resins and their cytotoxic potencies reported.