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A S Rathore

Publications and source records attributed to A S Rathore.

At least 19 recordsLinked to original sources
PubMed2001

Validatibility of a capillary isoelectric focusing method for impurity quantitation.

A strategy is presented for examining the validatability of a capillary isoelectric focusing (cIEF) method, intended for quantitation of product-related impurities in a protein drug substance, according to guidelines published by the International Conference on Harmonization (ICH). The results of this study demonstrate the suitability of cIEF as an analytical method for the quantitation of two product-related impurities in a protein drug substance: a monodeamidated degradation product and an aggregated form of the parent molecule. A range of impurity levels was generated by spiking the isolated impurity species, into a representative production lot of the drug substance. Six impurity spike levels (0.5-12% impurity for deamidated species and 0.5-8% impurity for aggregated species) were analyzed in triplicate. Measurement of impurity peak area percent in the spiked samples provided the data for computing specificity, accuracy, precision, linearity and limit of quantitation (LOQ) for the impurities. Accuracy, defined as the agreement of peak area percent for impurity species with the theoretical impurity percentage from the spike ratio, was 85-96% for the deamidated species and 73-97% for the aggregated species. A linear relationship was found between the measured area percent and the theoretical percent impurity for both impurity species (coefficient of determination, r2=0.9994 for deamidated species and =0.9827 for aggregated species). Precision (repeatability) studies demonstrated a low relative standard deviation (RSD) value (<6%) at all spike levels for both impurity species. Intermediate precision and reproducibility were evaluated by simulating many of the multivariable testing conditions expected during the life cycle of an analytical method, such as multiple equipment and laboratories. Repeated analyses of the drug substance under these varied conditions, yielded RSD values of <20%, for both impurity species. The LOQ, defined as the lowest impurity level where both accuracy and precision were achieved, was assigned at the 0.5% impurity level for both impurity species. This work illustrates a successful strategy in applying the ICH validation guidelines for impurity analytical methods to a cIEF method. Moreover, the data demonstrate the ability of cIEF to be used reliably as an analytical method for impurity quantitation.

Drug Contamination↗
PubMed2001

Enhancement of electroosmotic flow in capillary electrochromatography.

A major impediment to enhancing the speed of separation in capillary electrochromatography (CEC) is the upper limit on the electroosmotic flow (EOF) velocity by the maximal zeta potential of the chromatographic surface. Here, a new approach to speeding up EOF, suggested by Yang and El Rassi (Electrophoresis 1999, 20,18-23), is examined critically. It entails the use of a tandem arrangement of a separating column and an auxiliary column, the sole function of which is to boost EOF velocity in the separating column and thus facilitate faster analysis by CEC. Based on the principle of conservation of mass and current and using experimental data obtained in a wide range of conditions, the flow velocities in the separating and auxiliary columns were evaluated. The results show that an equidiameter open tubular auxiliary column offers a greater enhancement of EOF velocity than a packed column. Nevertheless, within the scope of the experiments the enhancement of EOF velocity by as much as 50% by using open tubular auxiliary columns has been obtained.

Chromatography↗
PubMed1999

Electrosmotic mobility and conductivity in columns for capillary electrochromatography.

Columns employed so far in capillary electrochromatography (CEC) contain both a packed and an open segment with concomitant changes of the electric field strength and the flow velocity at the interface of the two segments in such duplex columns. To take this into account in measuring, processing, and interpreting CEC data, a framework is presented for the evaluation of the conductivity ratio and the interstitial electrosmotic flow (EOF) mobility and their usage as tools for characterizing CEC columns. This is illustrated by experimental data obtained from measurement of the current and the EOF in capillary columns packed with different stationary phases. The current data yielded the ratio of the conductivities of the packed and open segments that has been shown to be useful for the evaluation of the porosity and tortuosity. It is assumed that these important packing characteristics are the same for the flow of current and for the flow of the bulk mobile phase in the CEC column. The EOF mobility in such duplex columns is defined in two different ways. The apparent mobility, which is widely reported at present, is obtained from the length of packed segment, the migration time, and the overall electric field strength. On the other hand, the actual mobility is obtained after taking into account the porosity and tortuosity of the packing as well. Thus, the actual mobility is made independent of the porosity and tortuosity and therefore can be useful to estimate the zeta potential for characterizing the packing surface. Measurements of both the apparent and actual electrosmotic mobilities for a number of different columns have shown that the apparent and actual mobilities are significantly different in their magnitude. For this reason, it is recommended that, instead of the apparent EOF mobility, the actual mobility is used for the characterization of the packing in CEC columns.

Chromatography, Liquid↗
PubMed1998

Effect of a predetection open segment in the column on speed and selectivity in capillary electrochromatography.

Columns in capillary electrochromatography (CEC) most commonly have the detection window located immediately after the retaining frit of the packed segment. Here, the properties of "duplex" columns having a predetection open segment between the frit and the detector window are examined with particular regard to the effect of the relative lengths of the packed and open segments on the separation of mixtures containing neutral and charged components. This configuration allows the use of columns with short packed segments in contemporary instruments for rapid separations. It is shown that, by varying the length of the packed segment, the balance of chromatographic and electrophoretic forces can be shifted, and the selectivity can be adjusted if the separation involves the interplay of both mechanisms. Expressions are presented for estimating the retention time in a duplex column if the chromatographic and electrophoretic properties of the sample components are known. The results are expected to facilitate CEC method development in selection of the respective column segment lengths for optimum separation.

Chromatography, Liquid↗
PubMed1998

Axial nonuniformities and flow in columns for capillary electrochromatography.

Unlike in capillary zone electrophoresis and microscale high-performance liquid chromatography, columns in capillary electrochromatography (CEC) have discontinuities of the electric field strength and the flow velocity at the interface of the packed and open segments of the column. The goal of the present work is to offer a framework for measuring and interpreting the relevant electrochromatographic parameters such as the electric field strength, the potential drop, and the flow velocity in the packed segment of a CEC column. This would help us gain further insight in the electrochromatographic process and facilitate the design of CEC separation technology and comparison of data from different sources. First, the flow of ions that is governed by the conservation of current is analyzed and the potential drop across the packed and open segments of the column calculated. Then, conservation of volumetric flow rate is used to calculate the flow velocities through the two segments and, further, to estimate the net flow velocity through such a column. To satisfy the mass conservation law, in most cases a "flow-equalizing intersegmental pressure", which is different from the pressures at the two ends of the column, develops at the interface of the packed and the open segments. The intersegmental pressure, induced to equalize the flow rates in the two segments, has been shown to have a significant effect on the magnitude as well as the radial distribution of the flow velocity in the open segment, where the net flow becomes a mixture of electroosmotic and pressure-driven flows.

Chromatography, High Pressure Liquid↗
PubMed1998

Cyclodextrin aided separation of peptides and proteins by capillary zone electrophoresis.

Carboxymethylated-beta-cyclodextrin (CMBCD) in the electrophoretic medium (aqueous 50 mM sodium phosphate, pH 2.5) enhanced the separation using raw fused-silica capillaries in CZE of the four standard proteins: alpha-chymotrypsinogen A, cytochrome c, lysozyme and ribonuclease A. Furthermore, with 20 mM CMBCD in the electrophoretic medium, the cis-trans isomers of angiotensin could be separated at room temperature, whereas the separation of the conformers required subambient temperatures as low as -20 degrees C without CMBCD in the electrophoretic medium [50 mM sodium phosphate (pH 2.5), containing 10% (v/v) methanol]. Addition of heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) had no effect on the separation of the above proteins and peptides. The results suggest that in microcolumn separation techniques, certain cyclodextrin additives can be useful selectivity enhancers.

Amino Acid Sequence↗
PubMed1998

Cyclodextrins as selectivity enhancers in capillary zone electrophoresis of proteins.

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including alpha-chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and beta-lactoglobulins A and B, was altered by adding 6-monodeoxy-6-monoamino-beta-cyclodextrin or carboxymethylated beta-cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6-di-O-methyl)-beta-cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of beta-cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.

Adsorption↗
PubMed1998

Periodic syphilis profile in a New Delhi hospital.

The study reports the prevalence of syphilis determined at intervals of ten years amongst attendees of the Sexually Transmitted Diseases Centre (STD), in New Delhi, from 1954, to 1994. It was observed that the number of STD cases increased by about eight times in 1984 as compared to 1954. The prevalence of STDs had risen from 5.5 per cent in 1964 to 14.7 per cent in 1994. However, the syphilis load has been decreasing since 1974. Childhood syphilis has decreased from 12.6 per cent in 1954 to 0.5 per cent in 1994. However in adult males it increased from 62.3 per cent in 1954 to 80.4 per cent in 1984. Cardiovascular syphilis was last reported in 1954. The prevalence of syphilis was approximately three times higher in males than in females.

Adult↗
PubMed1997

Displacement chromatography with on-column isomerization.

Displacement chromatography was simulated for the separation of two feed components interconverting by a reversible first order reaction and with Langmuirian adsorption behavior. The study was prompted by recent interest in the isolation of cis and trans forms of peptides containing one or more peptidyl-proline residues when the isomerization reaction interferes with the separation. The parameter values used in the simulations are similar to those found experimentally by reversed-phase chromatography and capillary electrophoresis of phenylalanine-proline dipeptide. From the concentration profiles computed by the finite difference scheme, the dependence of both the yield and production rate on the temperature, column length, flow velocity and displacer concentration was evaluated. The most important operational variable of the system is the temperature as it affects both the kinetic and adsorption parameters. The yield and production rate of the component of interest were evaluated as a function of the column length and displacer concentration under conditions that facilitate its efficient separation and the plots show an optimum. Nonetheless, optimal conditions for yield and production rate were considerably different. In the temperature range from 2 to 42 degrees C, the yield always decreases with increasing temperatures and for all the cases, optimum yield by displacement mandates the use of conditions such as pH, solvent and temperature under which the rate of interconversion is reduced to a level where it does not palpably interfere with the separation. On the other hand, under certain conditions optimal production rate can be obtained at higher temperatures.

Chromatography, High Pressure Liquid↗
PubMed1997

Capillary electrochromatography: theories on electroosmotic flow in porous media.

In view of the present interest in capillary electrochromatography (CEC), theories dealing with electroosmotic flow (EOF) in porous media are reviewed with particular regard to the use of packed capillaries in CEC. Two of the models found in the pertinent literature are applicable to CEC and give simple analytical solutions. The first of the two models is based on von Smoluchowski's work as adapted and extended by Overbeek. It deals with EOF through packed capillaries under conditions of low electric field strength where the EOF varies linearly with the field strength because there is no polarization of the double layer. Overbeek's model originally developed for porous media of infinite dimensions was modified in an attempt to account for the wall effect that assumes importance in the packed capillary columns used in CEC. The second model proposed by Dukhin and his coworkers predicts EOF of at least an order of magnitude higher than that expected by classical theories. This "electroosmosis of the second kind" is believed to occur in columns packed with conductive particles like ion exchangers at high electric field strengths when the double layer is polarized and the EOF becomes a non-linear function of the applied voltage. Conditions necessary for electroosmosis of the the second kind are likely to arise upon the further development of CEC when further enhancement of the speed of analysis is brought about at electric field strength higher than that employed at present.

Electric Conductivity↗
PubMed1997

Capillary zone electrophoresis of interconverting cis-trans conformers of peptidyl-proline dipeptides: estimation of the kinetic parameters.

Peptides containing proline residues, except at the N-terminus, exist in cis and trans forms due to the rigidity of the peptidyl-proline bond. Computer-simulated and experimental electropherograms have been used to study the interplay of the electrophoretic migration and the kinetics of the cis-trans interconversion. The measure of the interference by the reaction is the dimensionless Damköhler number (Da) which is the ratio of the migrant's residence time to the characteristic time of reaction in the capillary. The fraction of the trans conformer that is not separable with 90% purity is employed to quantify the extent of overlap between the concentration profiles of the two interconverting migrants and the effect of the various operational variables was examined by simulation. The deterioration of separation, as measured by the degree of overlap, is a quasi sigmoidal function of Da with the selectivity and the intrinsic efficiency of the system as the parameters. At sufficiently low temperatures the interference by reaction kinetics may vanish and the two conformers are separated. At high enough temperatures, when the rate of interconversion is very fast, the two conformers are not separated at all. A simple and rapid method is proposed for estimation of kinetic parameters for the cis-trans isomerization on the basis of data obtained with phenylalanyl-proline dipeptide. The procedure involves determination of the equilibrium constant by measuring equilibrium concentrations of the two conformers using nuclear magnetic resonance (NMR) or capillary zone electrophoresis (CZE) at very low temperature, correlation of the peak shapes in the electropherograms with the Da, and finally, evaluation of the forward rate constants from the assigned Da values in the domain: 0.01 < Da < 0.5. Separations using CZE were performed in the temperature range of 1-40 degrees C by using the Beckman P/ACE unit equipped with an auxiliary cooling system. The kinetic data thus obtained showed good agreement (average error less than 5%) with those measured by NMR.

Computer Simulation↗
PubMed1997

Domiciliary health counselling in patients with sexually transmitted diseases.

To improve treatment compliance, follow-up and attendance of STD patients and their contacts, a programme, based on domiciliary visits was attempted. The programme was limited to male defaulters not responding to written and telephone calls. House visits to 79 out of 169 defaulters were made. Approximately half the number of houses were found locked. Fourteen defaulting patients reported for completion of their treatment and follow-up. Of these 11 were directly interviewed. None of the 14 patients agreed to disclose the addresses of their extramarital partners. During the study period 51 female sex contacts reported for treatment as compared to 27 in the control group. Though the numbers were small the study revealed that direct contact with the patient yielded the best results. It is suggested that to make the system cost-effective the health workers should spend time with STD patients in the clinic to mark out the correct location and time for meeting the patient either in the work place or at home in case of a follow-up.

Aftercare↗
PubMed1996

Kinetic study on the cis-trans isomerization of peptidyl-proline dipeptides.

The equilibrium and kinetic parameters of cis-trans interconversion of dipeptides containing peptidyl-proline moiety were investigated using the in-column incubation method with both CZE and HPLC and the ad hoc dissolution method. The use of the latter was possible because the conformational make-up of the solid peptides, and consequently of their ad hoc solution, was sufficiently different from that of the solution at equilibrium. This method with 1H-NMR and CZE analyses yielded very similar results for the cis-trans isomerization of Phe Pro in aqueous sodium borate, pH 8.4, at 10 degrees C with an average value of 0.34 and 6.6 x 10(-5) s-1 for the equilibrium and rate constant, respectively. The in-column incubation method is performed by CZE or HPLC so that the conformers are separated in the first half of the column and then incubated in column where they interconvert and reach equilibrium. Subsequent separation in the second half of the column yielded four peaks. Thus by measuring the conformer composition as a function of the reaction time, the rate constant can be evaluated. The forward rate constant of 1.42 x 10(-4) s-1 determined by the CZE in-column incubation method for Phe-Pro isomerization at 10 degrees C was twice of the value obtained by the ad hoc dissolution method. It is believed that the inner wall of fused-silica capillaries has a catalytic effect on the isomerization. Computer simulation was also employed to gain further insight on the catalytic activity of the capillary inner wall on such isomerization. Whereas the experimental and simulation profiles of Phe-Pro in aqueous borate buffer, pH 8.4, with a 37 cm long capillary were in excellent agreement, a four times faster interconversion rate had to be used to match the experimental profile obtained with a 57 cm long capillary under otherwise identical conditions. The catalytic effect of the octadecyl silica stationary phase on the isomerization was confirmed by the in-column incubation method with HPLC. The overall rate of the cis-trans isomerization of Phe-Pro, which entails the reaction both on the stationary phase and in the mobility phase, was about six times faster at 0 degree C than the rate measured by NMR in free solution using the mobile phase containing 65% (v/v) sodium phosphate, pH 6.5, and 35% (v/v) methanol. The results presented here serve as a caveat that the effect of the wall in CZE or the stationary phase in HPLC on the reaction cannot be ignored.

Chromatography, High Pressure Liquid↗
PubMed1996

Separation parameters via virtual migration distances in high-performance liquid chromatography, capillary zone electrophoresis and electrokinetic chromatography.

Among the various differential migration processes of separation, high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) have emerged as the two major high-performance analytical techniques for separation of complex biological substances. In both HPLC and CZE with electroendoosmotic flow, the differential migration process can be divided into a separative component, which involves selective interactions with the stationary phase or differences in the electrophoretic migration velocities, and a non-separative component representing migration by convection that does not contribute directly to separation. The introduction of virtual migration distances leads to an additivity relationship for the two components that is applicable to both of the above techniques and facilitates the recognition of the underlying similarities as well as the expression and comparison of the various separation parameters. Examination of the key migration parameters led to the characterization and the classification of the various modes of CZE with electroendoosmotic flow. The treatment was extended to the analysis of capillary electrochromatography and micellar electrokinetic chromatography; two hybrid processes which exhibit features borrowed from HPLC and CZE. The use of virtual migration distances also led to a consistent and unified description of the characteristic parameters of these separation systems.

Chromatography, High Pressure Liquid↗