Rapid and sensitive estimation of isoniazid, pyrazinamide and rifampicin in combined dosage form by reversed-phase liquid chromatography

A rapid and sensitive method of estimation for isoniazid, pyrazinamide and rifampicin from pharmaceutical dosage was developed. The mobile phase consisting of 80:20, methanol-tetrabutyl ammonium hydroxide (0.005N) pH 3.0 adjusted with phosphoric acid was used with an ODS -CN bonded phase column. The separation of the above drugs was accomplished within 10 min at a flow rate of 1.5 ml/min. For accurate quantitation clofazimine was used as an internal standard with UV detector set at 265 nm. No interference from a variety of excipients present in the dosage forms was observed.     

Estimation of ipratropium bromide from aerosols by reversed-phase liquid chromatography

Abstract

Ipratropium bromide, a derivative of N-isopropyl noratropine, was estimated from the samples of aerosols. An isocratic, reversed-phase liquid chromatographic separation method was developed by using an ODS, 5 μm column with the mobile phase acetonitrile-sodium dihydrogen orthophosphate (0.05M)-diethylamine (50 : 50 : 0.1, v/v) pH adjusted to 4.5 with phosphoric acid. Recoveries obtained were in the range 98% to 102% of ipratropium bromide from aerosol. Fluoxetine hydrochloride was used as an internal standard for quantitation.     

Estimation of ipratropium bromide from aerosols by reversed-phase liquid chromatography

Ipratropium bromide, a derivative of N-isopropyl noratropine, was estimated from the samples of aerosols. An isocratic, reversed-phase liquid chromatographic separation method was developed by using an ODS, 5 μm column with the mobile phase acetonitrile-sodium dihydrogen orthophosphate (0.05M)-diethylamine (50 : 50 : 0.1, v/v) pH adjusted to 4.5 with phosphoric acid. Recoveries obtained were in the range 98% to 102% of ipratropium bromide from aerosol. Fluoxetine hydrochloride was used as an internal standard for quantitation.     

Determination of rifampicin and isoniazid in pharmaceutical formulations by HPLC

Abstract

A liquid chromatographic procedure for the analysis of Rifampicin (RIF) and Isoniazid (INH) in pharmaceutical dosage forms utilizing reverse phase chromatography was developed. Isolation of analytes was carried out under isocratic conditions with a octadecylsilane column and an aqueous mobile phase containing Methanol (75%) and 0.02 M Disodium Hydrogen Orthophosphate (25%) with pH 4.5 adjusted with orthophosphoric acid. The detection was done at 254 nm.

The method is unique in analysing Rifampicin precisely in combination with Isoniazid particularly in liquid formulation. The method is specific and can distinctly isolate the degradation product in suspension.     

Determination of rifampicin and isoniazid in pharmaceutical formulations by HPLC

A liquid chromatographic procedure for the analysis of Rifampicin (RIF) and Isoniazid (INH) in pharmaceutical dosage forms utilizing reverse phase chromatography was developed. Isolation of analytes was carried out under isocratic conditions with a octadecylsilane column and an aqueous mobile phase containing Methanol (75%) and 0.02 M Disodium Hydrogen Orthophosphate (25%) with pH 4.5 adjusted with orthophosphoric acid. The detection was done at 254 nm.

The method is unique in analysing Rifampicin precisely in combination with Isoniazid particularly in liquid formulation. The method is specific and can distinctly isolate the degradation product in suspension.     

Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

Two-dimensional polyacrylmide gel electrophoresis (2D-PAGE), perhaps the most widely used method in proteomics research, is often limited by sensitivity and throughput. Capillary isoelectric focusing (CIEF) coupled with electrospray ionization (ESI) mass spectrometry (MS) provides a liquid-based alternative to 2D-PAGE that can overcome these problems but is limited by ampholyte interference and signal quenching in ESI-MS. Inserting a reversed-phase liquid chromatography (RPLC) step between CIEF and MS can remove this interference. In this work, a CIEF-RPLC-MS system is described for separation and characterization of proteins in complex mixtures. CIEF is performed with a microdialysis membrane-based cathodic cell that also permits protein fractions to be collected, washed to remove ampholyte, and analyzed by RPLC-MS. CIEF performance with this cell is equivalent to that achieved with a conventional cathodic cell, and no loss of protein is observed during faction collection. The cell can be easily and safely retrofitted into commercial instrumentation and is applicable for peptide analysis as well. Protein detection at the low-femtomole level is demonstrated with little or no interference from ampholyte, and CIEF-RPLC-MS data are used to construct a plot of pI vs MW for a protein mixture. The current instrumental configuration allows seven fractions in the pI range 3-10 to be analyzed by RPLC-MS in 2 h.     

Rapid liquid chromatographic determination of paracetamol and diclofenac sodium from a combined pharmaceutical dosage

A simple, rapid reversed-phase HPLC method for the estimation of paracetamol and diclofenac sodium simultaneously in a combined dosage was developed. A -CN bonded phase columb was used with a mobile phase methanol-potassium dihydrogen phosphate (0.05M), (45:55) pH 3.5 adjusted with phosphoric acid at flow rate of 1.0 ml/min. For accurate quanittaiton, diltiazem hydrochloride was used as an internal standard at 270 nm.     

Molecular-level comparison of alkylsilane and polar-embedded reversed-phase liquid chromatography systems

Stationary phases with embedded polar groups possess several advantages over conventional alkylsilane phases, such as reduced peak tailing, enhanced selectivity for specific functional groups, and the ability to use a highly aqueous mobile phase. To gain a deeper understanding of the retentive properties of these reversed-phase packings, molecular simulations were carried out for three different stationary phases in contact with mobile phases of various water/methanol ratios. Two polar-embedded phases were modeled, namely, amide and ether containing, and compared to a conventional octadecylsilane phase. The simulations show that, due to specific hydrogen bond interactions, the polar-embedded phases take up significantly more solvent and are more ordered than their alkyl counterparts. Alkane and alcohol probe solutes indicate that the polar-embedded phases are less retentive than alkyl phases for nonpolar species, whereas polar species are more retained by them due to hydrogen bonding with the embedded groups and the increased amount of solvent within the stationary phase. This leads to a significant reduction of the free-energy barrier for the transfer of polar species from the mobile phase to residual silanols, and this reduced barrier provides a possible explanation for reduced peak tailing.     

Intact protein profiling of Chlorobium tepidum by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

Capillary isoelectric focusing (CIEF) coupled with reversed-phase liquid chromatography (RPLC) and electrospray ionization (ESI) mass spectrometry (MS) is shown to provide a liquid-based alternative to 2D-PAGE for intact protein profiling. This combination exhibits high resolution, sensitivity and throughput for protein profiling based on pI vs MW. The CIEF-RPLC-MS system described here facilitates the use of IEF markers for internal calibration of pI. It also provides a high dynamic range as evidenced by the detection of 100 pg (3 fmol) of a test protein spiked into 1 microg of a complex protein mixture. About 1200 individual proteins/polypeptides were detected from lysates of the green sulfur bacterium Chlorobium tepidum in a single <8 h run. The pI vs MW profile obtained from CIEF-RPLC-MS compares favorably with theoretical data derived from the C. tepidum genome and experimental data obtained from 2D-PAGE.     

Thermodynamic and kinetic characterization of polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography

The retention of six polycyclic aromatic hydrocarbons (PAHs) was characterized by reversed-phase liquid chromatography. The PAHs were detected by laser-induced fluorescence at four points along an optically transparent capillary column. The profiles were characterized in space and time using an exponentially modified Gaussian equation. The resulting parameters were used to calculate the retention factors, as well as the concomitant changes in molar enthalpy and molar volume, for each PAH on monomeric (2.7 micromol/m2) and polymeric (5.4 micromol/m2) octadecylsilica. The changes in molar enthalpy become more exothermic as ring number increases and as annelation structure becomes less condensed. The changes in molar volume become more negative as ring number increases for the planar PAHs, but are positive for the nonplanar solutes. In addition, the rate constants, as well as the concomitant activation enthalpy and activation volume, are calculated for the first time. The kinetic data demonstrate that many of the PAHs exhibit very fast transitions between the mobile and stationary phases. The transition state is very high in energy, and the activation enthalpies and volumes become greater as ring number increases and as annelation structure becomes less condensed. The changes in thermodynamic and kinetic behavior are much more pronounced for the polymeric phase than for the monomeric phase.     

Analysis of antimycin A by reversed-phase liquid chromatography/nuclear magnetic resonance spectrometry

A mixture of closely related streptomyces fermentation products, antimycin A, is separated, and the components are identified by using reversed-phase high-performance liquid chromatography with directly linked 400-MHz proton nuclear magnetic resonance detection. Analyses of mixtures of three amino acids, alanine, glycine, and valine, are used to determine optimal measurement conditions. Sensitivity increases of as much as a factor of 3 are achieved, at the expense of some loss in chromatographic resolution, by use of an 80-microL NMR cell, instead of a smaller 14-microL cell. Analysis of the antimycin A mixture, using the optimal analytical high-performance liquid chromatography/nuclear magnetic resonance conditions, reveals it to consist of at least 10 closely related components.     

高效液相色谱法快速测定水中的苯并(a)芘

本文建立了一种水中苯并(a)芘的快速测定方法。以环己烷萃取,过滤,浓缩,定容,用反相HPLC测定,方法的检出限为0.0025μg/L,RSD(n=8)为2.9%,加标回收率为98.9%—106.9%。     

Retention mechanisms in reversed-phase liquid chromatography. Stationary-phase bonding density and partitioning

The partitioning model of retention for reversed-phase liquid chromatography, described by mean-field statistical thermodynamic theory, asserts that one principal driving force for solute retention is the creation of a solute-sized cavity in the stationary phase. Beyond a critical stationary phase bonding density, increased grafted chain density should result in enhanced chain ordering, which will increase the energy necessary for solute cavity formation and result in decreased chromatographic partition coefficients. We have evaluated chromatographic partition coefficients over an octadecyl bonding density range of 1.6-4.1 mumol/m2 and have found a maximum in partition coefficient at approximately 3.1 mumol/m2. Retention, however, approximately plateaus due to compensating changes in the partition coefficient and stationary phase volume. This provides unequivocal evidence that partitioning is the dominant form of retention for small nonpolar solutes.     

Synthesis and evaluation of an aromatic polymer-coated zirconia for reversed-phase liquid chromatography

We synthesized a novel aromatic polymer-coated zirconia-based RPLC stationary phase by chemical adsorption of a copolymer of chloromethylstyrene and diethoxymethylvinylsilane onto zirconia (CMS/VMS-ZrO2). Characterization of the pore structure of the support by nitrogen porosimetry and inverse size-exclusion chromatography indicates that CMS/VMS-ZrO2 maintains the well-defined pore structure of the base material. Flow studies show that CMS/VMS-ZrO2 has good mass transfer characteristics. The reversed-phase retention characteristics of the new support are comparable to those of conventional silica-bonded phases. We have also evaluated the mechanical, thermal, and pH stability of CMS/VMS-ZrO2. The results show that CMS/VMS-ZrO2 is stable over a very wide range of pH (pH = 1-13) and at temperatures as high as 160 degrees C. Chromatographic separations of some low molecular weight aromatic analytes on CMS/VMS-ZrO2 and octadecyl-bonded silica phases indicate that there are some subtle but significant differences in the chromatographic selectivity of these two types of phases.     

Elucidation of carotenoid patterns in citrus products by means of comprehensive normal-phase x reversed-phase liquid chromatography

A novel approach for carotenoid analysis has been developed. Orange essential oil and juice carotenoids were separated by means of comprehensive dual-gradient elution HPLC, using normal phase with a microbore silica column in the first dimension (first D), reversed phase with a monolithic C18 column in the second dimension (second D), and a 10-port switching valve as an interface. An on-line photodiode array detector was used in order to obtain absorption spectra. Peak identification was obtained by combining retention data with the UV-visible spectra.     

Internal-surface reversed-phase silica support for direct injection determination of drugs in biological fluids by liquid chromatography

A new internal-surface reversed-phase (ISRP) silica support has been designed for direct injection analysis of drugs in biological fluids by liquid chromatography. The support, prepared by using a new enzyme, polymyxin acylase, has N-octanoylaminopropyl phases, bound only to the internal surfaces of the porous silica, and N-(2,3-dihydroxypropyl)-aminopropyl phases on the external surfaces in order to be nonadsorptive to proteins. The average pore diameter of the prepared ISRP silica support was 50 A, which is small enough to exclude macromolecules such as serum proteins from the pores. The new ISRP support can be used for the direct injection analysis of hydrophilic and hydrophobic drugs in serum or plasma without destructive accumulation of proteins over the eluent pH range of 3 to 7. The recovery of drugs from serum was almost 100%, regardless of the difference in their protein bindings.     

Measurement of mobile-phase volume in reversed-phase liquid chromatography and evaluation of the composition of liquid layer formed by solvation of packing materials

The mobile-phase volumes (Vm) in reversed-phase liquid chromatography (RPLC) with alkyl-bonded silica, defined as the difference between the total volume of eluent in the column (V0) and the volume of the eluent solvent layer formed by solvation of the bonded phase (VL), are determined by the method derived from the eluent electrolyte effect on the retention of ionic analytes. The validity of the Vm values obtained is evaluated by comparing them with the retention volumes of various organic compounds and inorganic ions, which have been suggested as unretained markers, and those obtained from a linear dependence of the logarithmic retention factor on the carbon numbers of homologous series. From the results obtained, it has been concluded that the solvated liquid phase on a column packing material should be assigned to a part of the stationary phase and the method developed for determination of the Vm value based on the ion partition model gives the most reasonable value as the mobile-phase volume in RPLC. The volume and the solvent composition of the solvated liquid phase on C1, C8, and C18 bonded silica are estimated, and the effects of organic modifiers and the physicochemical structures of the packing materials on these values are discussed.     

Thermodynamic interpretation of retention equilibrium in reversed-phase liquid chromatography based on enthalpy-entropy compensation

The fundamentals of the retention equilibrium in reversed-phase liquid chromatography (RPLC) are studied on the basis of enthalpy-entropy compensation (EEC). First, retention data were acquired and the influence of the nature of the compounds, organic solvent modifier, and temperature on these data was assessed. Then, the data were analyzed according to the four different methods proposed by Krug et al., and an EEC was formally established. Linear correlations were observed between the logarithm of the adsorption equilibrium constants under the different RPLC conditions, suggesting linear free energy relationships (LFERs). Finally, the variations of the retentions with the experimental conditions are shown to be quantitatively explained by a new model based on EEC. This model affords a comprehensive interpretation of the variations of retention originating from changes of either one parameter alone or several simultaneously. The slope and intercept of the LFER that relates two equilibrium systems are accounted for by the new model. The parameters of this model are the changes of enthalpy and entropy associated with the retention, the compensation temperatures, and the experimental conditions.