Fluorimetric liquid chromatographic analysis of amantadine in urine and pharmaceutical formulation

A simple and sensitive liquid chromatographic method is described for the analysis of amantadine and memantine. The method is based on the derivatization of amantadine and memantine extracted from alkalified samples with (2-naphthoxy)acetyl chloride at mild conditions. The resulting derivatives were analyzed by isocratic HPLC with a fluorimetric detector (lambdaex, 227 nm; lambdaem, 348 nm). The linear range for the determination of amantadine or memantine spiked in urine (1.0 ml) was 1.0-10.0 nmol with a detection limit of about 0.2 nmol (S/N = 3; injected sample 20 microl). Only amantadine preparations are available on our local market, and application of the method to the analysis of amantadine in formulation and in the urine of a dosed subject was demonstrated and proved feasible. Quantitation of AT in tablets or capsules is capable in the linear range of 2.0-50.0 microM. Toluene was used as the solvent for extracting amantadine or memantine in samples and the resulting toluene extract was directly subjected to subsequent derivatization without solvent replacement leading to a simpler analytical procedure.     

Comprehensive multi-dimensional liquid chromatographic separation in biomedical and pharmaceutical analysis: a review

'Multi-dimensional' liquid separations have a history almost as long as chromatography. In multi-dimensional chromatography the sample is subjected to more than one separation mechanism; each mechanism is considered an independent separation dimension. The separations can be carried out either offline via fraction collection, or directly coupled online. Early multi-dimensional separations using combinations of paper chromatography, electrophoresis and gels, in both planar and columnar modes are reviewed. Developments in HPLC have increased the number of measurable analytes in ever more complex matrices, and this has led to the concept of 'global metabolite profiling'. This review focuses on the theory and practice of modern 'comprehensive' multi-dimensional liquid chromatography when applied to biomedical and pharmaceutical analysis.     

High performance liquid chromatographic analysis of dehydroepiandrosterone and its pharmaceutical tablet formulation

A rapid, sensitive and stability indicating high performance liquid chromatographic method was developed and validated for the analysis of dehydroepiandrosterone (DHEA) in pharmaceutical tablet formulation. The analysis was done on a Supelcosil C(18) column (25 cm x 4.6 mm i.d., 5 microm). The mobile phase consisted of methanol:sodium acetate buffer solution (5 g/L):acetic acid (500 mL/L), 57:42:1, v/v/v, adjusted to pH 5 at a flow rate of 1 mL/min. Detection was carried out at a wavelength of 258 nm. The polynomial regression data for the calibration curve showed good linear relationship in the concentration range of 0.2-1 mg/mL with r = 0.9996. The method was validated for precision, accuracy and recovery. The limit of detection was found to be 50 ng/ microL. The method was applied for the analysis of DHEA in its pharmaceutical tablet formulation. The effects of different buffers and alcohols on the retention of DHEA were studied and the role of acetic acid as an organic phase modifier was also investigated.     

High-performance liquid chromatographic determination of amphotericin B in a liposomal pharmaceutical product and validation of the assay

A validated high-performance liquid chromatographic method is presented to quantitate amphotericin B (AB) in a liposomal pharmaceutical formulation. The analysis is based on the chromatographic separation of AB and 1-amino-4-nitronaphthalene (the internal standard) on a C18 muBondapac reversed-phase column with a mobile phase consisting of a mixture of acetonitrile and 0.02 M ethylenediamine tetra-acetic acid disodium salt at pH 5.0 (45:55, v/v). The chromatographic analysis time is less than 10 min, and the validation of the assay shows that it is selective, accurate, and linear for the concentration range of 2.50 to 7.50 microg/mL with a detection limit of 0.00500 microg/mL. The within-day and between-day relative standard deviation values are 1.26% (n = 18) and 1.25% (n = 8), respectively. The method described conforms to the validation of compendial methods used for finished pharmaceutical products in general and offers a reliable, quick, and cost-effective procedure for examining the consistency or quality-control analysis of AB in liposomal products. It can also be applied for the determination of AB in other nonliposomal lipid-based drug delivery systems that are on the market.     

Simultaneous microemulsion liquid chromatographic analysis of fat-soluble vitamins in pharmaceutical formulations: Optimization using genetic algorithm

An environmentally benign and simple method has been proposed for separation and determination of fat-soluble vitamins using isocratic microemulsion liquid chromatography. Optimization of parameters affecting the separation selectivity and efficiency including surfactant concentration, percent of cosurfactant (1-butanol), and percent of organic oily solvent (diethyl ether), temperature and pH were performed simultaneously using genetic algorithm method. A new software package, MLR-GA, was developed for this purpose. The results indicated that 73.6 mM sodium dodecyl sulfate, 13.64% (v/v) 1-butanol, 0.48% (v/v) diethyl ether, column temperature of 32.5 °C and 0.02 M phosphate buffer of pH 6.99 are the best conditions for separation of fat-soluble vitamins. At the optimized conditions, the calibration plots for the vitamins were obtained and detection limits (1.06–3.69 μg mL⁻¹), accuracy (recoveries > 94.3), precision (RSD < 3.96) and linearity (0.01–10 mg mL⁻¹) were estimated. Finally, the amount of vitamins in multivitamin syrup and a sample of fish oil capsule were determined. The results showed a good agreement with those reported by manufactures.     

Gas chromatographic determination of parabens in various pharmaceutical dosage forms

Summary A specific, sensitive and general applicabla gas chromatographic method is described for the determination of parabens in various liquid pharmaceutical preparations: propylparaben and butylparaben in a liquid antacid dosage form (I); methylparaben, ethylparaben and propylparaben in a syrup (II); methylparaben and propylparaben in a solution for injection (III). Each time one of the parabens is used as internal standard. The parabens are extracted with diethylether and derivatized by silylation. Different columns are used for the analysis of the parabens: 3% SE-30 column, a 3% QF-1 column for different selectivity, a 2% OV-1 column for isothermal operation.Special attention is attributed to the standard: the parabens are dissolved in a minimal amount of 0.1 M sodium hydroxide and extracted in the same way as the pharmaceutical dosage form.     

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. III. Method validation

The most important steps in the validation of high-performance liquid chromatographic (HPLC) methods are discussed. The establishment of system suitability data and the assessment of peak purity are demonstrated on the example of bisquaternary amino steroids. For the recognition of incomplete resolution of adjacent peak pairs, the absorbance-ratio method in which the ratio of absorbances at two preselected wavelengths are plotted as a function of time in combination with the separation of sample components subjected to various chemical and physico-chemical treatments (stress conditions) is applied. The separation power and performance of the HPLC systems are characterized by the system resolution (SR) and system selectivity (SS). The special demands of stability-indicating methods are summarized.     

High-performance liquid chromatographic determination of polysorbate 80 in pharmaceutical suspensions

A quick HPLC method is reported for the analysis of polysorbate 80 in pharmaceutical suspensions. A typical pharmaceutical suspension was mixed with dilute potassium hydroxide, and heated at 40 degrees C for 6 h. This procedure resulted in quantitative hydrolysis of polysorbate 80 to release oleic acid. A quick HPLC procedure was used to analyze the hydrolyzed samples without further sample treatment. Polysorbate 80 USP, treated in the same way as the pharmaceutical suspensions, was used as standard. Full validation tests were carried out and the validation studies demonstrated that this method is suitable for accurate and reproducible analysis of polysorbate 80 in pharmaceutical suspensions.     

High-performance liquid chromatographic analysis of emetine after oxidative activation to a fluorescent product

A clinically useful analytical method is described for monitoring plasma levels of emetine. The drug is initially extracted from plasma with dichloromethane (0.3 volumes). The extract can be analyzed directly by paired-ion reversed-phase high-performance liquid chromatography to levels of 500 ng/ml of plasma by spectrophotometric monitoring of column effluent. For analysis of emetine at lower concentrations, the dichloromethane extracts are subjected to mild mercuric acetate oxidation prior to separation, thereby converting emetine to a fluorescent product. Spectrofluorometric monitoring of the column effluent readily extends the sensitivity of the assay to 10 ng of emetine/ml of plasma. At these levels measurements can be made with a precision of +/- 4%.     

High performance liquid chromatographic determination of butamyrate citrate in pharmaceutical preparation

An HPLC method was developed and validated for the determination of butamyrate citrate. The HPLC separation was achieved on a diol column (300 × 4.6 mm) packed with 5.0 μm particle size using a mobile phase of ammonium acetate buffer (pH = 6.5) and methanol (750:250, v/v) at a flow rate of 1.4 ml min^?1. The UV detector was operated at 225 nm. The method was validated for specificity, linearity, precision, accuracy and robustness. The retention time was 5.9 min. The proposed method provided linear responses within the concentration range 75–225 μg ml^?1 with LOD and LOQ values of 0.69 and 2.29 μg ml^?1, respectively. Correlation coefficient (r) of the regression equation was 0.9999. The method was found to be precise, accurate, and reproducible.     

Simultaneous liquid chromatographic determination of ezetimibe and simvastatin in pharmaceutical products

A reversed-phase liquid chromatographic (LC) method was developed and validated for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical dosage forms. The LC method was carried out on a Synergi fusion C18 column (150 mm x 4.6 mm id) maintained at 45 degrees C. The mobile phase consisted of phosphate buffer 0.03 M, pH 4.5-acetonitrile (35 + 65, v/v) run at a flow rate of 0.6 mL/min, and detection was made using a photodiode array detector at 234 nm. The chromatographic separation was obtained within 15.0 min, and calibration graphs were linear in the concentration range of 0.5-200 microg/mL. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated, giving results within the acceptable range for both compounds. Moreover, the proposed method was successfully applied for the routine quality control analysis of pharmaceutical products.     

Measurement uncertainty of liquid chromatographic analyses visualized by Ishikawa diagrams

Ishikawa, or cause-and-effect diagrams, help to visualize the parameters that influence a chromatographic analysis. Therefore, they facilitate the set up of the uncertainty budget of the analysis, which can then be expressed in mathematical form. If the uncertainty is calculated as the Gaussian sum of all uncertainty parameters, it is necessary to quantitate them all, a task that is usually not practical. The other possible approach is to use the intermediate precision as a base for the uncertainty calculation. In this case, it is at least necessary to consider the uncertainty of the purity of the reference material in addition to the precision data. The Ishikawa diagram is then very simple, and so is the uncertainty calculation. This advantage is given by the loss of information about the parameters that influence the measurement uncertainty.     

Comparison of various liquid chromatographic methods for the analysis of avermectin residues in citrus fruits

Various liquid chromatographic (LC) techniques for analyzing avermectin (Abamectin) were compared after extraction of residues from citrus fruit samples by matrix solid-phase dispersion (MSPD). LC with UV and fluorescence detection were used as also was LC coupled to the mass spectrometer by an electrospray interface. The results obtained by the three methods were compared in terms of sensitivity and selectivity. The combination of MSPD extraction and LC with fluorescence detection have made it possible to quantify 0.5 microg kg(-1) of Abamectin in 0.5 g of orange sample, with an overall average recovery of 94%. The procedure provides a simple and sensitive method for monitoring Abamectin residues in citrus fruit at the levels required by legislation.     

Simple fluorimetric liquid chromatographic method for the analysis of undecylenic acid and zinc undecylenate in pharmaceutical preparations

A simple and selective liquid chromatographic method is described for the analysis of undecylenic acid (UA) and zinc undecylenate (ZnUA) in pharmaceutical preparations. The method is based on the derivatization of the analytes extracted from various samples with 2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate. The resulting derivative was analyzed by liquid chromatography with fluorimetric detection. The quantitation of the method is in the range of 3.0-50.0 microM UA with a detection limit of about 0.3 microM (S/N = 3 with 10 microl injection). We found that acetonitrile is a selective solvent for differentially dissolving UA from coexisted ZnUA in compound formulation. This results in the specific analysis of UA in the presence of ZnUA and simply analyzing the coexisted ZnUA by the value of total UA (UA+ZnUA) minus that of UA. Application of the method to the analysis of undecylenic acid and zinc undecylenate in ointment, powder and solution preparations proved feasible.     

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. IV. Selection of most applicable separation system

Different analytical tasks in the pharmaceutical analysis can be classified according to the separation problems into three main groups: trace analysis, assay methods and separation of closely related compounds including isomers. The most important requirements of high-performance liquid chromatographic (HPLC) methods with respect of the separation problems are summarized. Considerations and recommendations for the selection of the most applicable HPLC system to solve particular analytical problems are discussed. HPLC methods can be compared on the basis of the system resolution (SR) and system selectivity (SS). Criteria developed for the characterization of HPLC methods considering the difficulties created by the different analytical problems are established. The principles of the selection of the most applicable separation systems are demonstrated through some practical examples in pharmaceutical analysis.