A method for rapidly predicting drug tissue distribution using surfactant vesicle electrokinetic chromatography

Lung tissue distribution of an inhaled drug is important for its potency in the airways and with minimum systemic effects within its dose range. As the lung has the smallest diffusion distance of all the organs in the body and negligible diffusion delays, the characteristics of drug distribution in the lung will mainly depend on drug binding to both tissue and plasma protein. This research aims to develop and evaluate surfactant vesicle electrokinetic chromatography (SEKC) methods for high throughput profile prediction of tissue distribution for inhaled drugs. Several electrokinetic chromatography methods reported in the literature, as well as immobilised artificial membrane chromatography, were compared and evaluated in respect to chromatographic characteristics and statistical correlations. Among these methods, the docusate sodium salt (AOT) SEKC system showed good reproducibility, short run time, and the highest selectivity for alkylphenone test compounds. It also showed a significant statistical correlation between the retention of inhaled drugs and their in vivo volume of distribution at steady-state (V(ss)) in whole human body neglecting the plasma protein-binding differences. Stronger correlations were observed between the AOT SEKC retention of a series of basic drugs and their rat lung tissue-to-plasma water partitioning coefficient (K(pu)), which is affected only by drug binding to the tissue constituent. Further, on comparing correlations between AOT SEKC retention and K(pu) at various rat tissues, it was observed that the strongest correlation was with lung tissue distribution, while the weakest was with brain tissue distribution.     

Comparing micellar electrokinetic chromatography and microemulsion electrokinetic chromatography for the analysis of preservatives in pharmaceutical and cosmetic products

In this study, separation and determination of nine preservatives ranging from hydrophilic to hydrophobic properties, which are commonly used as additives in various pharmaceutical and cosmetic products, by micellar electrokinetic chromatograpy (MEKC) and microemulsion electrokinetic chromatography (MEEKC) were compared. The effect of temperature, buffer pH, and concentration of surfactant on separation were examined. In MEKC, the separation resolution of preservatives improved markedly by changing the sodium dodecyl sulfate concentration. Temperature and pH of running buffers were used mainly to shorten the magnitude of separation time. However, in order to detect all preservatives in a single run in a MEEKC system, a microemulsion of higher pH was needed. The separation resolution was improved dramatically by changing temperature, and a higher concentration of SDS was necessary for maintaining a stable microemulsion solution, therefore the separation of the nine preservatives in MEEKC took longer than in MEKC. An optimum MEKC method for separation of the nine preservatives was obtained within 9.0 min with a running buffer of pH 9.0 containing 20 mM SDS at 25 degrees C. A separation with baseline resolution was also obtained within 16 min using a microemulsion of pH 9.5 which composed of SDS, 1-butanol, and octane, and a shorter capillary column at 34 degrees C. Finally, the developed MEKC and MEEKC methods determined successfully preservatives in various cosmetic and pharmaceutical products.     

Systematic development and validation of stability indicating micellar electrokinetic chromatography methods for early stage drug candidates

Suitable methods for assessment of purity, potency and stability of new drug substances and drug products are required to be rapidly developed and validated to provide appropriate data for early project development decisions. In order to routinely provide methods of consistent and suitable quality to meet increasingly aggressive timelines, systematic approaches to both develop and validate analytical technologies have been developed. Systematic approaches to evaluate separation parameters such as buffer pH, buffer ionic strength, surfactant concentration, organic modifiers, organic modifier concentration, applied voltage and temperature were evaluated for an early stage drug candidate. Techniques to improve method precision and ruggedness were also examined. Finally, the validation results from the micellar electrokinetic chromatography method utilizing an internal standard were compared against the simultaneously developed high-performance liquid chromatography method.     

Successful spectrofluorometric and chemiluminescence methods for the estimation of azathioprine as an immunosuppressive drug in pharmaceutical preparation

The determination of azathioprine (AZA), a mercaptopurine derivative, has attracted a particular attention in most researches. Herein, spectrofluorometric (I) and chemiluminescence (II) methods were successfully confirmed to estimate azathioprine (AZA) in bulk powder form and pharmaceutical preparation. The optimum conditions to improve the fluorescence and chemiluminescence intensity of AZA were investigated. The method (I) measured the native fluorescence intensity of AZA in methanol as solvent, without any addition. The micelle-enhanced fluorescence of AZA was affected by the surfactant addition, type of solvent, pH value, and the volume of sodium dodecyl sulfate (SDS). The method (II) consisted in the improvement of the weak signal of calcein–KMnO₄ chemiluminescence system by introducing synthesized silver nanoparticles (AgNPs) in the presence of AZA. The analytical curves were linear in the concentrations range 5.0 × 10⁻⁸ − 1.0 × 10⁻⁴ M and 5.0 × 10⁻⁹ − 2.0 × 10⁻³ M with a detection limit equal to 1.5 × 10⁻⁹ M and 2.6 × 10 ⁻¹⁰ M for techniques (I) and (II), respectively. The statistical analysis indicated no significant difference between the proposed and reported methods. The suggested techniques were successfully used to determine azathioprine in its tablet form and were validated according to ICH guidelines.     

Investigation of pharmaceutical high-performance liquid chromatography assay bias using experimental design

This article presents a systematic approach to investigate, document, and eliminate pharmaceutical HPLC assay bias using experimental design. This is the first article to describe the application of experimental design in the area of assay bias. It is found that both formulation and analytical variables can contribute to pharmaceutical HPLC assay bias using model compounds and formulations.     

Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry

Studying the permeability of compounds across a Caco-2 cell monolayer is an established in vitro model to screen for oral absorption and to evaluate the mechanism of transport. This assay can also be used to evaluate compounds as potential P-glycoprotein substrates and/or inhibitors. The traditional methods of sample analysis (high-performance liquid chromatography (HPLC) with a UV or fluorescence detector) limit the throughput and sensitivity of this assay. Data are presented here describing the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the analysis of samples derived from the Caco-2 cell studies. During the analysis an automatic switching valve was used to divert the flow from the HPLC column to waste for the first minute, preventing the early eluting salts from entering and contaminating the LC/MS interface. This approach allows the rapid and accurate determination of drug transport across the Caco-2 cell monolayer. The high sensitivity and specificity of LC/MS/MS make this technique an ideal candidate for the low concentration and high throughput routine analysis of Caco-2 cell solutions, especially if multiple compounds are administered and analyzed simultaneously. Thus, the use of LC/MS/MS will increase the value of the Caco-2 cell assay as an in vitro screening tool.     

Derivatizing assay for the determination of aldehydes using micellar electrokinetic chromatography

In this work, the use of a novel derivatization agent for the determination of aldehydes (in this particular case: formaldehyde, acetaldehyde, propionaldehyde, and valeraldehyde) using micellar electrokinetic chromatography is reported. The derivatization reaction is based on the reaction of aldehydes with benzhydrazide to form the corresponding derivates with maximum absorbance at 250 nm. The experimental conditions of the derivatization reaction as well of the separation were optimized. The adducts were separated with a +22 kV voltage at a temperature of 29°C. The adducts’ separation was performed in less than 14 min using as the running buffer a mixture containing 110 mmol/L of sodium dodecyl sulfate and 27 mmol/L of sodium tetraborate at pH 9.45. Samples were injected using hydrodynamic mode (50 mbar × 5 s). The calibration curves were linear up to 15.0 mg/L with r ² above 0.99. Intra and inter‐day precisions were in average 3 and 4%, respectively, and recoveries were in average of 95%. Limits of detection and quantification were around 0.5 and 1.5 mg/L, respectively. The developed method was successfully applied in the analysis of low molar weight aldehydes in yogurt and vinegar samples.     

Cationic double-chained surfactant as pseudostationary phase in micellar electrokinetic capillary chromatography for drug separations

The cationic double-chained surfactant didodecyldimethylammonium bromide (DDAB) was used as pseudostationary phase (PSP) in micellar electrokinetic capillary chromatography (MEKC). Its performance on the three kinds of drugs, i.e., basic, acidic, and neutral drugs, was systematically investigated. Nicotine, cotinine, caffeine, lidocaine, and procaine were selected as the model basic drugs. Good baseline separation and high efficiency were obtained under the optimal separation condition that consisted of 50mM phosphate (pH 4.0) and 0.08 mM DDAB. Three basic phenylenediamine isomers can also be well separated with DDAB in buffer. In addition, DDAB can form cationic bilayer on the capillary wall, thus the wall adsorption of basic analytes was greatly suppressed. Compared with commonly used CTAB, the separation of basic drugs was significantly improved with a much lower amount of DDAB present in the buffer. The DDAB-involved MEKC also showed superiority to CTAB upon the separation of acidic drugs, amoxicillin and ampicillin. In the case of neutral compounds, a good separation of resorcinol, 1-naphthol and 2-naphthol was achieved with 0.1mM DDAB and 30% (v/v) acetonitrile (ACN) present in buffer. Hence, it was concluded that the double-chained cationic surfactant DDAB can be a good substitute for traditional single-chained surfactant CTAB in MEKC.     

Implementation of a rapid and automated high performance liquid chromatography method development strategy for pharmaceutical drug candidates

A comprehensive, fully automated strategy is demonstrated for HPLC-UV chromatographic method development using ChromSword optimization software. The strategy involves: (1) the automated screening of various column and mobile phase combinations, (2) rational selection of the best starting conditions; and (3) subsequent automated method development to generate optimized separation methods. Pharmaceutical compounds were applied to solve problematic drug impurity separations. ChromSword software automates the screening, optimization, and documentation steps thus reducing the method development time. The strategy was compared to a manual method development approach showing the automated method strategy affords better selectivity in a shorter time.     

Comparison of microemulsion electrokinetic chromatography and micellar electrokinetic chromatography as methods for the analysis of ten benzophenones

Microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic chromatograpy (MEKC) were compared for their abilities to separate and detect ten similar benzophenones, which are commonly used as UV filters in various plastic and cosmetic products. Sodium dodecyl sulfate (SDS) concentration and column temperature rarely affected separation resolution for MEEKC, but separation of benzophenones could be improved by changing the SDS concentration and column temperature for MEKC. Buffer pH and ethanol (organic modifier) were found to markedly influence the separation selectivity for both MEEKC and MEKC systems. In addition, a higher electric voltage improved the separation efficiency without a noticeable reduction in separation resolution for MEEKC, whereas it caused a poor separation resolution for the MEKC system.     

Micellar electrokinetic capillary chromatography as an alternative method for determination of hydrocortisone and its most important associated compounds in local pharmaceutical preparations

Summary A new, simple, and accurate micellar electrokinetic chromatographic (MEKC) method is described for quantification of hydrocortisone, hydrocortisone hemisuccinate, hydrocortisone acetate, mystatin, oxytetracycline, Zn-bacitracin, polymyxin B, and lidocaine in ocular and cutaneous pharmaceutical products. The separation was performed at 25°C and 25 kV, with 15mm phosphate +15mm borate buffer, pH 8.2, and 60mm sodium dodecylsulfate (SDS) in 10∶1 (%,v/v) methanol-water as background electrolyte. Under these conditions the analysis time is approximately 23 min. The method has been used for quantification of these compounds in different commercial pharmaceutical products and gave good results when compared with reference spectrophotometric and HPLC methods.     

Evaluation of a multiarray system for pharmaceutical analysis by microemulsion electrokinetic chromatography

A multiplexed capillary electrophoresis (CE) system equipped with 96 channels was evaluated for high-throughput screening in drug discovery by microemulsion electrokinetic chromatography (MEEKC). Method transfer from a single channel to a multichannel CE system is described. Loss of efficiency and reduced migration times could be elucidated to the poor efficacy in Joule heat dissipation by forced air cooling in the multiarray system compared to liquid cooling in the single channel instrument. On the other hand, only 48 channels could actually be used because of the maximum total current of 3 mA. Precision data remained below 8% and 9% for migration times and peak areas, respectively. Some UV-detector cross-talk interference between neighboring capillary channels was noted. Impurities at 0.5% compared to the main peak (100%) could be detected with the multiplexed system which is 10 times lower compared to the single capillary system. Higher efficiency and improved figures of merit (absolute sensitivity and no cross-talk interferences) were obtained by using an array of only 24 capillaries.     

Determination of piribedil in pharmaceutical formulations by micellar electrokinetic capillary chromatography

A fast and simple micellar electrokinetic capillary chromatographic method was developed for the analysis of piribedil in pharmaceutical formulations. The effects of buffer concentration, buffer pH, sodium dodecyl sulphate (SDS) concentration, organic modifier, applied voltage and injection time were investigated. Optimum results were obtained with a 50 mM borate buffer at pH 8.0 containing 50 mM SDS by using a fused silica capillary (50 m internal diameter, 72 cm effective length). The sample was injected hydrodynamically for 4 s at 50 mbar pressure and the applied voltage was +30 kV. The detection wavelength was set at 205 nm. Diflunisal was used as an internal standard. The analysis was performed at 25 °C and the total run time was 14 min. The method was suitably validated with respect to linearity range, limit of detection and quantification, precision, accuracy, specificity and robustness. The linear calibration range was 5–100 g mL^–1 and the limit of detection was determined as 1 g mL^–1. The method developed was successfully applied to the determination of piribedil in pharmaceutical formulations. The results were compared with a spectrophotometric method reported in the literature and no significant difference was found statistically.     

Enantiomeric purity methods for three pharmaceutical compounds by electrokinetic capillary chromatography utilizing highly sulfated-gamma-cyclodextrin as the chiral selector

Methods for enantiomeric purity by electrokinetic chromatography were developed and validated for three pharmaceutical compounds, each utilizing highly sulfated-gamma-cyclodextrin (HS-gamma-CD) as the chiral recognition agent. Two of the compounds are weak bases, hence charged at low pH, and the third is a quaternary nitrogen compound, charged at all pH. In each instance quantification was via an authentic reference standard with addition of an internal standard. Separation was on a 61 cm x 50 microm untreated capillary under reverse polarity with a background electrolyte of 5% HS-gamma-CD in pH 2.50 lithium phosphate buffer. Each method was validated with respect to the usual validation parameters, notably recovery and precision, yielding results, including limits of detection and quantitation, that allow reporting the minor enantiomer to 0.1% and less. In applying the methods, all batches of bulk drug tested were shown to be of enantiomeric purity > or =99.9%.     

High performance liquid chromatography assay for piroxicam in pharmaceutical products

A high performance liquid chromatographic assay for piroxicam in pharmaceutical preparations is described. The method uses a reversed-phase C18 column with pH 3 aqueous buffer/methanol, 55:45, v/v mobile phase, and is selective for piroxicam in the presence of other "oxicams," synthetic precursors, by-products, degradation products, metabolites, and related compounds. Applications to capsules, tablets, ointments, suppositories, ophthalmic suspensions, and rodent feeds are cited.     

Micellar electrokinetic chromatography assay for the simultaneous quantification of urinary transferrin and albumin

A micellar electrokinetic chromatography (MEKC) method for simultaneous determination of urinary transferrin and albumin has been developed. The performance of the assay was evaluated. Under the optimum conditions, the linearity of transferrin and albumin is in the range of 10~1000 mg L^?1. The detection limits are 1.3 mg L^?1 for transferrin and 1.1 mg L^?1 for albumin, with relative standard deviations of less than 5.4%. The within-run and between-run coefficients of variation were <5.5% for transferrin and <8.2% for albumin, respectively. The recoveries of transferrin and albumin are 97.7~106.1%. The performance of the MEKC assay was compared with radioimmunoassay, and correlated well with this.     

Micellar electrokinetic capillary chromatography as an alternative method for the determination of ethinylestradiol and levo-norgestrel

A method for quantifying of ethinylestradiol (ETE) and levo-norgestrel (LEV) in pharmaceutical products by micellar electrokinetic chromatography (MEKC) is described. The separation was carried out at 25 degrees C and 25 kV, using a 20 mM borate buffer (pH 9.2), 15 mM sodium dodecylsulfate (SDS) in 30% acetonitrile/water (v/v). Under these conditions the analysis takes about 7 min. The method has been applied for quantifying both compounds in six different commercial contraceptives and the proposed method gave good results when compared with a reference liquid chromatographic (LC) method.     

Application of micellar electrokinetic chromatography to the determination of sultamicillin in oral pharmaceutical preparations

A micellar electrokinetic capillary electrophoretic method for determination of sultamicillin in Unasyn oral preparations--tablets and suspension--was evaluated. Phosphate-borate buffer at pH 7.0 containing 1.0% sodium dodecylsulfate was used as a mobile phase. The elaborated method ensures separation of sultamicillin from p-toluenesulfonic acid and the impurities, ampicillin, sulbactam and penicillamine. The method was validated for specificity, reproducibility, precision, accuracy and assay linearity (in a concentration range of sultamicillin of 0.05-1.5 mg/ml). Statistical analysis by Student's t-test showed no significant differences between the results obtained by micellar electrokinetic chromatography and HPLC, t(calculated) 0.519 for suspension assays and 0.284 for tablets assays were smaller then t(tabulated).