Direct injection analysis of hexamethylene bisacetamide in biological fluids by an HPLC column switching technique with a micellar mobile phase

Summary This paper describes the direct injection analysis of the anti-cancer drug hexamethylene bisacetamide (HMBA) in biological fluids by an HPLC column switching technique. The first chromatographic column, which provides for sample extraction and cleanup, employs a micellar mobile phase with SDS as the modifier. The second column, coupled on-line to the first, utilizes reversed-phase conditions for analysis. UV detection is employed at 210 nm. 282 samples from 12 cancer patients were analysed and good pharmacokinetics curves obtained. The drug gives recoveries of 94.0–100.9% with a relative standard deviation of 1.88%. A sample analysis is completed within 15 minutes. This method should be satisfactory not only for the analysis of HMBA but also, probably for other drugs in biological fluids.     

Use of micellar mobile phases for the chromatographic determination of melamine in dietetic supplements

Melamine is a nitrogen-rich industrial chemical which is occasionally used to increase the apparent protein content of different products destined for human and animal consumption. In this work, a liquid chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulfate (SDS) buffered at pH 3, a C18 column and UV detection is reported for the determination of melamine in dietetic supplements. Samples were reconstituted with a SDS solution and were directly injected, thus avoiding long extraction and experimental procedures. Melamine was eluted in less than 10 min with no interference by other compounds of the matrices. The optimum mobile phase composition was taken by a chemometrical approach that considers the retention factor, efficiency and peak shape. Validation was performed following the indications of the European Commission (Decision 2002/657/EC). The following parameters were considered: linearity (0.02-100 μg mL(-1); R(2) = 0.9996), intra- and inter-day precisions (<12.4%), accuracy (90.0-101.3%), and robustness (less than 9.8% and 5.1%, for retention time and peak area, respectively). The limits of detection and quantification were 9 and 20 ng mL(-1), respectively. Recoveries for several spiked samples were in the 85.8-114.3% range. These results indicate that the proposed methodology is useful for routine analysis of control quality of infant formula and adult dietetic supplements.     

Characterization of micellar mobile phases for reversed-phase chromatography

Cationic, anionic, and nonionic surfactants are characterized for their usefulness as micellar mobile phases in reversed-phase chromatography. Conditions found previously to provide optimum chromatographic efficiency for sodium dodecyl sulfate also provide high efficiency for the cationic and nonionic surfactants studied. The use of 3% n-propanol in the micellar mobile phase and column temperatures of 40°C appear to offer a broadly applicable solution to the low efficiency previously reported for micellar mobile phases. A chromatographic method for the determination of critical micelle concentrations is reported; it compares favorably with literature methods. Micellar mobile phases are shown to mimic ion-pairing mobile phases, allowing the separation of neutral solutes as well as solutes charged oppositely to the surfactant and offer a more rugged method of analysis than hydro-organic ion-pairing methods.     

Use of micellar mobile phases for the chromatographic determination of clorazepate, diazepam, and diltiazem in pharmaceuticals

An ODS-2 column, a micellar mobile phase of high elution strength containing 0.1M sodium dodecyl sulfate and 3% (v/v) butanol, and ultraviolet detection at 230 nm are used for the determination of either of two benzodiazepines (clorazepate and diazepam) and a benzothiazepine (diltiazem) in pharmaceuticals. The procedure is shown to be competitive against conventional chromatography with methanol-water mobile phases, especially for diltiazem. The composition of the micellar mobile phase is selected using a predictive strategy based on an accurate retention model and assisted by computer simulation. Calibration graphs are linear at least in the 2.5 to 20 microg/mL, 4 to 20 microg/mL, and 5 to 40 microg/mL ranges for clorazepate, diazepam, and diltiazem, respectively. The intra- and interday repeatabilities (%) are clorazepate (1.7, 5.2), diazepam (0.43, 3.7), and diltiazem (0.36, 3.1). Limits of detection are well below the concentrations of the drugs found in the commercial pharmaceutical preparations analyzed. The drug contents evaluated with the proposed procedure are compared with the declared contents given by the manufacturers. The achieved percentages of label claim are usually between 95 and 104%.     

The chromatographic quantification of hydrophobicity using micellar mobile phases

Summary In micellar liquid chromatography (MLC), the hydrophobicity of a compound is the predominant factor in its retention and interaction with micelles. A non-linear empirical model can describe the dependence between the retention factor (logk) in MLC and the logarithm of partition coefficients octanol-water (logP). An algorithm based on such a model has been used to makelogP predictions. Retention data for series of neutral compounds eluted with different mobile phases and alkyl-bonded stationary phases have been used to test the predictive ability of the algorithm. The results of this approach are compared with those obtained from automatic computational software packages.     

Chromatographic determination of caffeine in pharmaceutical formulations using micellar mobile phases

Summary An HPLC procedure is described for the determination of caffeine in pharmaceutical preparations. A Spherisorb octadecylsilane ODS-2 C₁₈ analytical column and spectrophotometric detection at 273 nm were used. The chromatographic behaviour of caffeine with different micellar eluents containing sodium dodecyl sulphate (SDS) is described. The determination of caffeine in pharmaceutical preparations was performed by use of a mobile phase containing 0.05 M sodium dodecylsulphate (SDS) and 1.5% propanol at pH7. At a 6.0 g mL^–1 concentration level the peak area and peak height repeatability were 2.6 and 2.4%, respectively. The application of the proposed method to the analysis of five pharmaceutical formulations, using peak heights as the dependent variable, gave recoveries between 85 and 104% of the values declared by the manufacturers. The proposed procedure for the determination of caffeine is rapid (15 min per sample), reliable and free from interferences.     

Use of column switching for the determination of niacinamide in compound feed

A high-performance liquid chromatographic method is described for the determination of niacinamide in compound feed. The niacinamide is extracted with 0.2 M hydrochloric acid in order to suppress the hydrolysis of the niacinamide to nicotinic acid. The chromatography is carried out with the aid of column switching. An RP-18 column is used for the preseparation, and the analytical separation takes place in a cation-exchange column. With the proposed method, the limit of determination is about 2 ppm of niacinamide.     

Comparision of varuious bulk fluids and modifiers as near-crtical mobile phases on a polymeric column using linear solvatio energy realationships

Linear solvation energy realationships (LSERS) were used to qunatitatively compare the relative contributions of dipolarity/polarizability, hydrogen bonding, and other types of intermolecular interactions to retention and selectivity using various bulk moblie phase component. Using experimental condition which differ only by the compostion of the bulk moble phase component, the factors which cause selctivity to differ between HPLC, subcritical fluid chromatography and supercritical fluid charomatography were decovoluted. Heptance-based HPLC mobile phases showed superior selectivities towards analytes which differ in hydrogen bond donating ability, gas-to-hexadecane partion coefficint and dipolarity/ploarizability. subcritical fluid chromatography with HFC-134a (1, 1, 1, 2-tetrafluoroethane) as the bulk fluid produced superior seclectivities for which differ in hydrogen bond accepting ability and execess molar refraction properties. Many of these factors showed temperature dependences which act to attenuate or accenture the particular intermolcular interaction.     

Liquid chromatography of some steroid hormones in aqueous-organic,micellar, and cyclodextrin mobile phases

The retention of hydrocortisone, progesterone, dexamethasone, and prednisolone is studied by HPLC and thin-layer chromatography using aqueous-organic, micellar, and cyclodextrin mobile phases. It is demonstrated that micellar and cyclodextrin mobile phases improve the efficiency and selectivity of the chromatographic separation of the indicated hormones compared to aqueous-organic eluents. By comparing the numbers of theoretical plates, the heights equivalent to a theoretical plate, and resolution it was found that micellar mobile phases are more efficient than cyclodextrin ones and can be used for work with Sorbfil plates.     

Use of a three-factor interpretive optimisation strategy in the development of an isocratic chromatographic procedure for the screening of diuretics in urine samples using micellar mobile phases

Screening of diuretics in urine is feasible through direct injection of the samples into the chromatographic system and isocratic reversed-phase liquid chromatography (RPLC) with micellar-organic mobile phases of sodium dodecyl sulfate (SDS) and 1-propanol. The surfactant coverage of the chromatographic column makes the addition of organic competing amines less necessary than in conventional aqueous-organic RPLC to achieve well-shaped peaks. Also, the range of elution strengths of micellar mobile phases required to elute mixtures of hydrophobic and hydrophilic diuretics is smaller. This allows the isocratic separation of the diuretics within adequate analysis times. An interpretive methodology is applied to optimise the resolution of a mixture of 15 diuretics of diverse polarity and acid-base behaviour (althiazide, amiloride, bendroflumethiazide, benzthiazide, bumetanide, canrenoic acid, chlorthalidone, ethacrynic acid, furosemide, piretanide, probenecid, torasemide, triamterene, trichloromethiazide and xipamide), using pH and concentrations of surfactant and organic modifier in the mobile phase as separation factors. Twelve diuretics were resolved in 25 min using 0.055 M SDS-6.0% 1-propanol at pH 3.0. The mixture of 15 diuretics was also resolved with two mobile phases showing complementary behaviour: 0.05 M SDS-5.6% 1-propanol at pH 5.4 and 0.11 M SDS-5.4% 1-propanol at pH 4.2. The results were applied to the analysis of urine samples with limits of detection similar to those usually reported for aqueous-organic RPLC, taking into account that the samples were injected without any previous treatment to separate or preconcentrate the analytes.     

Pre-column switching techniques for the determination of drugs and metabolites in body fluids in research and routine analysis

The use of a column switching system for direct injection of samples and of a sample clean-up on reversed phase pre-columns is described. The pre-columns were filled with spherical C-18 silica gel of particle size 30 microns. Two applications are reported on: (1) the direct injection of serum samples for the simultaneous analysis of nine antiepileptic drugs and metabolites and (2) the determination of phenytoin and of carbamazepine in serum ultra-filtrates. The purge liquid for the sample clean-up was diluted phosphoric acid, and the eluent mixture for the chromatographic separation was water/acetonitrile. The analytical column (length 12.5 cm) was filled with C-18 silica gel of particle size 5 micron. A gradient elution was chosen for the first application, while the second application was carried out using isocratic chromatographic conditions.     

Use of various filters for generating modified supercritical fluid mobile phases

Summary Adding various components to supercritical carbon dioxide in supercritical fluid chromatography can extend or significantly alter the solvating properties. Polar samples which are difficult to analyze with pure supercritical CO₂ because of their high polarity can be separated by addidng polar modifiers. In this paper, a new mixing method using an HPLC filter for adding polar modifier to CO₂ is described. Although several filters were tried, only one could keep the amount of modifier in the mobile phase constant for a long time. The amount of water or methanol dissolved in supercritical CO₂ was measured by an amperometric microsensor made of a thin film of perfluorosulfonate ionomer (PFSI).     

Use of low volatility mobile phases in electroosmotic thin-layer chromatography

A variant of electroosmotic thin-layer chromatography is suggested with the use of low volatility compounds as mobile phases aimed at drastically decreasing the evaporation of the mobile phase and improving the reproducibility of the method. The linear movement velocity of zones of separated compounds is experimentally shown to increase 2-12-fold in electroosmotic chromatography (compared to similar values in traditional TLC). The separation efficiency is also considerably increased.     

Development and validation of a method to determine amoxicillin in physiological fluids using micellar liquid chromatography

A simple and robust method was developed for the routine identification and quantification of amoxicillin by micellar LC. Amoxicillin, a beta-lactamase inhibitor, is one of the most commonly prescribed drugs in the treatment of urine and skin structure infections. In this work, amoxicillin was determined in urine samples without any pretreatment step in a phenyl column using a micellar mobile phase of 0.10 M SDS and 4% butanol at pH 3. A UV detection set at 210 nm was used. Amoxicillin was eluted at 5.1 min with no interference by the protein band or endogenous compounds. Linearities (r >0.9998), intra- and interday precisions were determined (RSD (%) 0.4-2.7% and 0.3-5%, respectively, in micellar media, and 0.14-2.6% and 0.13-6%, respectively, in urine), and robustness was studied in the method validation. LOD and LOQ were 0.04 and 0.1 microg/mL in micellar media and 0.14 and 0.34 microg/mL in urine, respectively. Recoveries in the urine matrix were in the range of 95-110%. The validated method proved to be reliable and sensitive for the determination of amoxicillin in urine samples.     

Analysis of omeprazole and its main metabolites by liquid chromatography using hybrid micellar mobile phases

Omeprazole is a selective inhibitor of gastric acid secretion and is one of the most widely prescribed drugs internationally. A chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulphate and propanol buffered at pH 7 and a C18 column is reported for the determination of omeprazole and its principal metabolites (omeprazole sulphone and hydroxyomeprazole) in urine and serum samples.In this work, direct injection and UV detection set at 305 nm was used. Omeprazole and its metabolites were eluted in less than 11 min with no interference by the protein band or endogenous compounds. Adequate resolution was obtained with a chemometric approach, in which the retention factor and shape of the chromatographic peaks were taken into account. The analytical parameters including linearity (r > 0.9998), intra- and inter-day precision (RSD, %: 0.6-7.9 and 0.14-4.7, respectively) and robustness were studied in the validation of the method for the three compounds. The limits of detection and quantification were less than 6 and 25 ng mL⁻¹, respectively. Recoveries in micellar medium, plasma and urine matrices were in the 98-102% range. Finally, the method was successfully applied to the determination of omeprazole and its metabolites in physiological samples. Omeprazole was also analysed in pharmaceutical formulations.     

Solvent‐free mixed micellar mobile phases: An advanced green chemistry approach for reversed‐phase HPLC determination of some antihypertensive drugs

Minimizing the amount of organic solvents without loss in chromatographic performance has been an important step toward greening analytical methodologies. Mobile‐phase composition is the key for maintaining separation efficiency in liquid chromatography while decreasing the procedure hazardousness. If sodium dodecyl sulfate is mixed with Brij‐35 in the mobile phase, they could be used as a green alternative for using organic modifiers. In this research, the effect of changing the relative amounts of both surfactants was studied on the chromatographic performance and separation efficiency of ten antihypertensive drugs belonging to different categories. The use of surfactants has many advantages including low cost and toxicity, safe environmental disposal, unique selectivity besides high solubilization capabilities. The optimum separation was maintained using a mobile phase (0.01 M Brij‐35, 0.08 M sodium dodecyl sulfate and 0.01 M sodium dihydrogen phosphate/pH 5) on reversed‐phase C18 core–shell column at flow rate 1.5 mL/min and temperature 30°C. The method was successfully applied for the determination of the drugs in various marketed dosage forms. International Conference of Harmonization guidelines were followed to validate the developed method. Additionally, the method was verified on the Green Analytical Procedure Index in regards to the greenness and found to be an excellent green alternative method.     

Modeling of the separation of two enantiomers using a microbore column

A microbore column packed with Chiralcel OB (cellulose tribenzoate coated silica) was used for the measurement of the single and competitive equilibrium-isotherm data of the 1-indanol enantiomers by frontal analysis. The amount of sample needed for the isotherm data acquisition was about 20 times less than that required with a conventional column. The data obtained were fitted to different single and competitive isotherm models. Both the single and the competitive data sets fitted best to the same Bilangmuir (BL) isotherm model with small differences in the numerical values of the parameters. The best fitted Bilangmuir single and competitive isotherm models were used to predict the overloaded experimental profiles of both pure enantiomers, of the racemic mixture, and of different enantiomeric mixtures. All the calculated profiles were in excellent agreement with the experimental ones. This agreement confirms that in many chiral separations, the competitive isotherms can be derived from data acquired from the mere racemic mixture with a sufficient accuracy for a correct prediction of the band profiles of all kinds of enantiomer mixtures, making possible the computer-assisted optimization of the experimental conditions.     

Preparation and characterization of lauryl methacrylate-based monolithic microbore column for reversed-phase liquid chromatography

Poly(lauryl methacrylate-co-ethylene dimethacrylate) monoliths were in situ synthesized within the confines of a silicosteel tubing of 1.02 mm i.d. and 1/16" o.d. for microbore reversed-phase HPLC. In order to obtain practically useful monoliths with adequate column efficiency, low flow resistance, and good mechanical strength, some parameters such as total monomer concentration (%T), cross-linking degree (%C) and polymerization temperature were optimized. High-efficiency monoliths were successfully obtained by thermal polymerization of a monomer mixture (40%T, 10%C) with a binary porogenic solvent consisting of 1-propanol and 1,4-butandiol (7:4, v/v) at a high temperature of 90 °C. The morphology and porous structure of the resulting monoliths were assessed by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC), while the column performance was evaluated through the separations of a series of alkylbenzenes in acetonitrile-water (50:50, v/v) eluent. At a normal flow rate of 50 μL/min (corresponding to 1.66 mm/s), the optimized monolithic columns typically exhibited theoretical plate numbers of 6000 plates/10 cm-long column for amylbenzene (k>40), and the pressure drop was always less than 1 MPa/10 cm. The monoliths, which were chemically anchored to the tube inner wall surface using a bifunctional silylation agent, exhibited adequate mechanical strength of up to 12-13 MPa, and were properly operated at 10 times higher flow rate than normal, reducing the separation time to one tenth. The lauryl methacrylate-based monolithic column was applied to a rapid and efficient separation of ten common proteins such as aprotinin, ribonuclease A, insulin, cytochrome c, trypsin, transferrin, conalbumin, myoglobin, β-amylase, and ovalbumin in the precipitation-redissolution mode. Using a linear CH(3)CN gradient elution at a flow rate of 500 μL/min (10-times higher flow rate), 10 proteins were baseline separated within 2 min.     

Imitation of artificial membrane system via mobile phases with Tween-80 and cholic acid in biopartitioning micellar chromatography

The chromatographic behaviour of compounds of biomedical significance was studied using micellar mobile phases modified with polyoxyethylene (20) sorbitan monooleate (Tween-80). The influence of the surfactant within the 0.75-4% concentration range on the retention factor of model compounds was investigated. The biological surfactant cholic acid was introduced into the mobile phases in order to approach to the structure of natural membranes, viz. erythrocyte and cytoplasmatic membranes. It was found that curves of dependence of retention factor vs concentration of Tween-80 in the absence and presence of cholic acid in the mobile phase considerably diverge with one another, especially in the 2-3% concentration range of Tween-80 using C18-type support. Increasing the concentration of Tween-80 resulted in the increase of retention factors using phenyl-coated stationary phase.