Gradient High-performance Liquid Chromatography Research Articles

Chemical composition distribution of graft copolymer prepared by macromonomer technique: Determination by gradient HPLC

AbstractThe chemical composition distributions (CCDs) of poly(methyl methacrylate)‐graft‐polystyrene samples prepared from the polystyrene macromonomers having methacryloyl and p‐vinylbenzyl end‐groups were determined by high‐performance liquid chromatography (HPLC) based on reversed‐phase and normal‐phase adsorption modes. The CCDs determined by both modes were in good agreement with one another, indicating that the effect of the molecular weight distribution on the CCD is negligible. The results demonstrated the features of the CCDs in agreement with the theoretical predictions and the strong effect of the intrinsic reactivity of the end‐group on the copolymerization reactivity of the macromonomer.

A rapid anion exchange high-performance liquid chromatography method for the measurement of HbA2 in whole blood.

We developed a binary gradient high-performance liquid chromatography (HPLC) method for measuring HbA2 in whole blood samples using a Pharmacia Mono Q column (1 mL) and measurement at 415 nm. The assay requires a simple lysis and centrifugation step before injection onto the column. We found good agreement of results between the HPLC method and the Helena column chromatography method. The within batch precision was 2-6% and between batch precision was 4-6%. We found that using 30 mM Tris buffers (pH 7-8) with a sodium chloride gradient resulted in short analysis times and good chromatographic separation of HbA2, HbS and HbA. We conclude that this is a robust assay for the diagnosis of beta-thalassaemia.

Analysis of mixtures containing free fatty acids and mono-, di- and triglycerides by high-performance liquid chromatography coupled with evaporative light-scattering detection

Commercial monostearates of glycerol, generally used as antistatic agents for thermoplastic polymers, consist of a complex mixture of mono-, di- and trisubstituted glycerides and the corresponding fatty acids. Thus far, the glycerides and the fatty acids have been analyzed separately by high-performance liquid chromatography (HPLC). In fact, the simultaneous analysis of glycerides and fatty acids requires esterification of the acids in order to avoid overlapping of the chromatographic peaks. This paper describes an HPLC method which allows the direct separation and identification, simultaneously and in a single run, of the variously substituted glycerides, and also the corresponding saturated fatty acids that are found as by-products in commercial glycerol monostearates. The procedure is based on the use of a ternary gradient HPLC instrument equipped with an evaporative light-scattering detector; the stationary phase was a reversed-phase RP-8 end-capped (Merck) column; the mobile phase was two consecutive binary gradients consisting of acetonitile-water plus acetic acid (0.1%, v/v) and acetonitrile-methylene chloride. The method is fast and shows high sensitivity and selectivity, being able to separate also the positional isomers of mono- and digycerides in addition to the mixed di- and triglycerides.

Plasma pharmacokinetics and urinary excretion of the polyamine analogue 1, 19-bis(ethylamino)-5, 10, 15-triazanonadecane in CD2F1 mice.

The pharmacokinetics of 1, 19-bis(ethylamino)-5, 10, 15-triazanonadecane (BE-4-4-4-4) were determined in CD2F1 female mice after administration of i.v. bolus doses of 20 mg/kg (approximately the dose lethal to 10% of the study animals, approximately LD10) as well as 15, 10, and 5 mg/kg and after s.c., i.p., or p.o. doses of 20 mg/kg. BE-4-4-4-4 in plasma and urine was derivatized with dansyl chloride and measured by gradient high-performance liquid chromatography (HPLC) with fluorescence detection. Data were modeled by noncompartmental and compartmental methods. The declines observed in plasma BE-4-4-4-4 concentrations after i.v. delivery of 20, 15, 10, and 5 mg/kg were modeled simultaneously using an interval of 2000 min between doses and were best approximated by a two-compartment, open, linear model. The time courses of plasma BE-4-4-4-4 concentrations after i.p. and s.c. delivery were fit best by a two-compartment, open, linear model with first-order absorption. Peak plasma concentrations of BE-4-4-4-4 measured following an i.v. dose of 20 mg/kg ranged between 30 and 33 microgram/ml, the terminal elimination half-life was 94 min, and the volume of distribution (Vdss) was 850 ml/kg. The plasma pharmacokinetics of BE-4-4-4-4 were linear with dose. BE-4-4-4-4 (0.5 and 2.0 microM) in mouse plasma was approximately 67% protein-bound. Bio-availabilities after i.p., s.c., and p.o. delivery were 40%, 50%, and approximately 3%, respectively. Urinary excretion of parent BE-4-4-4-4 in the first 24 h after dosing accounted for less than 30% of the delivered dose. As BE-4-4-4-4 proceeds toward and undergoes clinical evaluation, the data and analytical method presented herein should prove useful in formulating a dose-escalation strategy and, possibly, evaluating toxicities encountered.

Stepwise binary gradient high-performance liquid chromatographic system for routine drug monitoring

Drug therapy is usually optimized by concentration measurement in patient serum. High-performance liquid chromatography (HPLC) is one of the most important analytical techniques used for therapeutic drug monitoring (TDM) of drugs for which no immunoassay kits are available. HPLC has been frequently used for screening purposes in toxicology, too. The Merck Tox Screening System (MTSS) has been developed for the identification of substances by a combination of gradient HPLC with diode-array detection and identification with a database system. For routine TDM an isocratic HPLC system is more suitable because of shorter analysis time, better reproducibility of retention index and better precision of results. Therefore we defined a set of methods in steps of 10% of the two MTSS eluents. Three examples are shown: Amiodarone, Indometacine and Thiopental. New applications to test for other substances can be transferred to an isocratic system after a complete MTSS gradient run.

Polymer molecular weight characterization by temperature gradient high performance liquid chromatography

We report a novel method of temperature gradient interaction h.p.l.c. for the characterization of molecular weight distribution of macromolecules. A very fine and reproducible control of interaction between polymer chains and the packing material can be achieved by altering the column temperature. A mixture of 10 polystyrene standard samples (molecular weight range: 1700-2 890 000) of narrow molecular weight distribution are analysed by this method. Near complete separation down to the baseline is achieved with the use of a single C18 bonded silica column. This method is thus proven to provide a much superior resolution to the conventional size exclusion chromatography.

Degradation kinetics of growth hormone-releasing hexapeptide (GHRP-6) in aqueous solution

The degradation kinetics of growth hormone-releasing hexapeptide (GHRP-6) as a function of pH and temperature as well as buffer species and ionic strength has been studied by stability-indicating reversed-phase gradient high-performance liquid chromatography. Degradation chromatograms were confirmed in different pH solutions and the observed degradation reaction rates approximately followed first-order kinetics with respect to GHRP-6. The influence of the various buffer species (acetate, citrate, phosphate and borate) was shown to be different and the maximum stability of GHRP-6 was revealed to be in acetate buffer of pH 5.5–6.0. Degradation of GHRP-6 was greater in citrate-containing buffers than in acetate-containing ones. Furthermore, in the citrate-containing buffers, the higher buffer concentration caused greater degradation than the lower ones, but the concentration effect was negligible in acetate-containing buffers. Aqueous solution of GHRP-6 buffered with acetate (0.01 M, pH 5.5) showed a predicted t90% of 4.73 years at 20°C.

Stability Study of the Active Constituents in theCentella asiaticaExtract Formulations

The creams and dragees containing Centella asiatica plant extract were subjected to accelerated storage conditions, and stability of the active constituents, viz. asiatic acid, asiaticoside, madecassic acid, and madecassoside were evaluated, using reverse phase gradient high-performance liquid chromatography.

Solid-phase extraction of arginine vasotocin and isotocin in fish samples and subsequent gradient reversed-phase high-performance liquid chromatographic separation

Gradient high-performance liquid chromatography (HPLC) preceded by a solid-phase extraction (SPE) step is described for determining arginine vasotocin and isotocin, the neurohypophysial nonapeptides, in fish plasma samples. The combination of these two methods significantly improves the separation and increases the sensitivity of the assay. The proposed assay may be a useful alternative for analysis of similar nonapeptides in plasma without the use of radioisotopes, while taking into consideration a difference in detection sensitivity.

A rifampicin-induced hepatic microsomal enzyme system for the generation of cyclosporine metabolites

A drug-induced rabbit hepatic microsomal enzyme system has been developed to produce milligram quantities of cyclosporine metabolites (CMs). Using a rifampicin-induced microsomal preparation in the presence of a NADPH regenerating system, 60% of the cyclosporine (CsA) was converted to CMs in 2 h. The CMs were recovered by solid phase extraction, and separated by gradient high performance liquid chromatography with two Ultrasphere Ocyl (C8) columns connected in tandem. More than 20 CMs were resolved. The quantities of major CMs produced by 45 mg of microsomal proteins were established by comparing peak areas with known concentrations of authentic CM standards. These major CMs included AM1, AM9, AM19, AM4N, AM1c and the aldehydic isomers (AM1cAL plus AM1AL). Other CMs that were not quantified included AM14N, AM4N9, AM1A, AM1c9, and AM1D1. Several CMs remained to be identified. All CMs were detected by radioimmunoassay using a non-specific CsA antiserum. The purity of the CMs were confirmed by fast atomic bombardment mass spectrometry. Similar findings were observed when erythromycin or trolandomycin was used to induce the hepatic microsomal enzymes. The procedure used to generate CMs was simple. With the enzyme fraction derived from one rabbit liver, 90 to 100 mg of CMs can be obtained. In this study, the metabolite patterns of CsA produced by rabbit liver microsomes were shown to resemble those observed for humans. These results indicate the possibility of using rabbit models to predict CsA biotransformation in man. The CMs generated by this enzyme system can be used to acquire information relevant to the situation in man.

Separation of lipid classes from plant tissues by high performance liquid chromatography on chemically bonded stationary phases

AbstractThe wide range of lipid classes found in plant tissues, including simple lipids, glycolipids, and phospholipids, have been resolved by gradient high performance liquid chromatography with evaporative light‐scattering detection. The lipids from potato tubers were used to optimize the separations. Adsorption chromatography with silica gel gave disappointing results for the plant glycolipids. Much better separations were obtained with chemically bonded stationary phases, e.g. those with propionitrile or polymeric vinyl alcohol as the functional moiety. With careful calibration, good reproducibility was possible. Only highly acidic lipids such as phosphatidylserine still present some problems.

Preparation of Sample Solution Using Pronase Treatment for Determination of Food Coal-Tar Dyes in Foods

A method for preparation of sample solutions has been developed for determination of food coal-tar dyes in foods by using pronase. Twelve kinds of dyes were extracted with a mixture of 1% NH4OH and ethanol from foods which were hydrolyzed by pronase treatment. Then the extract was concentrated under vacuum, cooled in ice, treated with tetrabutylammonium hydroxide and cleaned up by using Sep-pak Plus tC18 cartridges. The dyes retained on the replaced cartridge were eluted with a mixture of methanol and tetrabutylammonium phosphate solution (pH 5.0), and determined by gradient high performance liquid chromatography with visible wavelength detectors.The recoveries of food coal-tar dyes were more than 76.0% from foods spiked at 1 and 10μg/g levels.

Predicting 1-octanol-water partition coefficient by high-performance liquid chromatography gradient elution

The method to predict 1-octanol-water partition coefficients (Kow) from capacity factors (k′) obtained by Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) has been extended to use gradient elution rather than isocratic elution. The mobile phase has been changed either linearly or exponentially with time. The initial composition of the mobile phase and its rate of change affected the log Kow versus log k′ relation. This relation was exponential in linear gradient experiments. For non-linear gradient elution in which the water fraction of the mobile phase decreased exponentially from 100% to approach 0% asymptotically, a physically-based equation describing the dependence of log Kow on log k′ has been derived. Without any preliminary estimation, RP-HPLC gradient elution allows a precise prediction of log Kow over a range of nearly six orders of magnitude.

Control of adsorption and solubility in gradient high-performance liquid chromatography

Copolymers from styrene and methyl methacrylate (MMA) were separated by both normal and reversedphase gradient chromatography. Both modes could be performed by sudden-transition gradients where the polymers were injected into a non-solvent whose polarity was either rather low (e.g.,n-heptane) or high (e.g., acetonitrile). Then the solvent strength of the starting eluent was rapidly increased to a given level by addition of dichloromethane. Under properly defined conditions, the sample components still remained on the column. Elution could be triggered off by the steady addition of another non-solvent whose polarity was opposite of that of the starting non-solvent. Thus, the mixture of five copolymers with MMA content ranging from 14 to 84% could be separated on a polar cyanopropyl column by injection inton-heptane and elution through acetonitrile (normal phase mode) and on a RP C18 column by injection into acetonitrile and elution throughn-heptane (reversed-phase mode), provided that in both modes about 30% dichloromethane was added to the starting non-solvent. The elution sequence in the reversed-phase mode was opposite to that in the normal-phase mode, obeying the approved polarity rules of chromatography in both cases.

Evaluation of liquid chromatography coupled with high-field 1H NMR spectroscopy for drug metabolite detection and characterization: the identification of paracetamol metabolites in urine and bile.

The applicability of coupled reversed-phase high performance liquid chromatography (HPLC)-NMR spectroscopy for the detection and identification of paracetamol (N-(4-hydroxyphenyl)acetamide) and its sulfate, glucuronide and N-acetylcysteinyl metabolites in the unprocessed biological fluids, human urine, rat urine and rat bile, is investigated. Analysis of these samples was performed by gradient HPLC elution and directly coupled 500 MHz 1H NMR spectroscopy detection using a combination of one- and two-dimensional NMR methods in stopped-flow mode. The stopped-flow approach is demonstrated to be an efficient technique for identification of drug metabolites which have, for example, a UV-chromophore. Stopped-flow HPLC analysis with NMR detection is a viable technique and halting the chromatographic process several times during a run has a negligible effect on the separation and NMR characterization. The post-acquisition data processing method of 'quantified maximum entropy' is shown to provide a means of improving the quality of spectra for minor components, thus aiding NMR resonance assignments.

A nonequilibrium radioimmunoassay for angiotensin II

A method for the measurement of angiotensin II levels in dog plasma is described. The method is similar to previously published assays in that it couples gradient high-performance liquid chromatography (HPLC) with radioimmunoassay (RIA) and requires blood sample collection and processing to plasma in the presence of protease inhibitors. The unique feature of the present method is that it utilized a commercially available angiotensin II RIA run under nonequilibrium conditions. Performing the angiotensin II RIA under nonequilibrium conditions increased RIA sensitivity to allow for a minimal detectable limit of 0.75 pg/mL, a limit of detection not achievable with current commercially available RIAs. This lower limit of detection will now allow for the measurement of circulating levels of angiotensin II. Quality control pools of dog plasma fortified with 4.59–50 pg/mL angiotensin II were assayed and analytical recoveries (ARs) and coefficients of variation (CV) of 72.2%–111% and 3.67%–19.0% were observed for the respective pools.

Separation of Polyethylene Glycol Oligomers on Normal-Phase and Reversed-Phase Materials by Gradient High Performance Liquid Chromatography and Detection by Evaporative Light Scattering. A Comparative Study

Abstract Separation of the polyethylene glycols PEG-200, PEG-300, PEG-600 and PEG-1000 by gradient high performance liquid chromatography is reported on a C18 as well as a Si 80 column in aqueous mixtures of acetonitrile, acetone and methanol as organic solvents. Detection was performed by means of evaporative light scattering. Further, the Mn and Mw values were determined by gel permeation chromatography and refractive index detection. Marked differences in the chromatographic behaviour occured between the two stationary phases with all three modifiers. Whereas PEG-200 and PEG-300 exhibited better peak resolution Rs on the C18 matrix, the inverse effect was observed with PEG-600 and PEG-1000, which reveal much better Rs on bare silica gel. Further, a dependence of the Rs of the PEG-600 and PEG-1000 samples (consisting of a substantially higher number of oligomers versus PEG-200 and PEG-300) from the organic modifier was seen, which increases in the range methanol <acetone <acetonitrile. A hypothetical vi...

Control of adsorption and solubility in gradient high performance liquid chromatography. Part 4. Sudden-transition gradient elution of styrene/ethyl methacrylate copolymers in reversed phase mode

Copolymers from styrene and ethyl methacrylate have been separated according to composition byn-heptane gradients on a C18 bonded phase after injection into acetonitrile and subsequent sudden transition to a concentration of either dichloromethane or tetrahydrofuran between 30 and 50% or 20 and 50%, respectively. Acetonitrile is a polar non-solvent for the copolymers under investigation and ensures proper retention of the samples on a non-polar stationary phase. Dichloromethane and tetrahydrofuran are good solvents of moderate polarity. The addition of, e.g., 30 vol% of one of these solvents increased the dissolution power of the starting eluent but not to the extent necessary for elution. The latter was achieved by the addition ofn-heptane, which is a non-solvent for the polymers investigated. Thus, its eluting power must be understood as the consequence of its modifying effect on the polarity of the eluent mixture. The higher the content of copolymer in ethyl methacrylate the earlier it was eluted. Since acetonitrile andn-heptane are only partly miscible, phase diagrams were measured after the addition of either tetrahydrofuran or dichloromethane as a third component. Homogeneous mixtures were obtained on addition of about 30% solvent (one of both of the latter).

Control of adsorption and solubility in gradient high performance liquid chromatography. Part 3. Sudden-transition gradient elution of styrene/ acrylonitrile copolymers

Control of adsorption and solubility in gradient high performance liquid chromatography. Part 3. Sudden-transition gradient elution of styrene/ acrylonitrile copolymers

A multidimensional approach to analysis of cerebrospinal fluid biogenic amines in schizophrenia: I. Comparisons with healthy control subjects and neuroleptic-treated/ unmedicated pairs analyses

Recent hypotheses and findings indicate that measurements of interactions between cerebrospinal fluid (CSF) biogenic amine systems, rather than measurement of CSF biogenic amine metabolites, better correlate with clinically important findings in schizophrenia. To test these hypotheses, we used a recent technological advance in high performance liquid chromatography with electrochemical detection and combined it with multivariate statistical analyses to study biogenic amine concentrations in CSF in schizophrenia. This approach enabled the study of the interactions of several metabolites of each of the three major neurotransmitter pathways (dopaminergic, noradrenergic, and serotonergic) to test existing hypotheses regarding the neurobiochemical basis of schizophrenia. Twenty biogenic amines, their metabolites, and other compounds from 24 medication-free schizophrenic patients and 12 normal control subjects were simultaneously measured using a recently developed technique of gradient high performance liquid chromatography coupled with a 16-channel electrochemical array detector. After covariation for storage time, results of a stepwise discriminant function analysis comparing the control and patient groups identified tryptophan, tryptophol, and epinephrine as discriminating variables. Hotelling's paired T 2 test from a subgroup of schizophrenic patients studied while they were and were not receiving neuroleptic treatment did not yield any significant differences between subgroups. A discussion of the findings and a comparison with previous studies of CSF biogenic amines in schizophrenia are presented.