Simultaneous determination of dexamethasone and betamethasone in pharmaceuticals by reversed-phase HPLC

Chromatographia - An HPLC method for the simultaneous determination of the glucocorticoids betamethasone and dexamethasone is described. The method based on the separation of these compounds using...     

Quantitative screening for steroids in animal feeding water using reversed phase LC with gradient elution

Several isocratic separations for the determination of 20 steroids (STER) in animal feeding water samples (AFWS) from drinking-trough by LC using a mobile phase ACN/H(2)O (35:65 v/v) and different RP columns (Hypersil C18, Gemini C18 (GM), Purospher Star C18, Synergi Max C12, and Synergi Fusion) and UV detection were obtained. The elution order was the same: a first group of corticoids (CC) was early eluted, a second group of CC and anabolics (AAS) exhibited intermediate retention, and a third group constituted by AAS was strongly retained. To improve the separation performances of the isocratic separations an ACN gradient elution optimization was carried out for each column. The most satisfactory results were obtained using a Purospher column which allowed the separation of 19 STER in an analysis time close to 26 min. After sample preparation using SPE, method validation was performed in an AFWS spiked with STER according to the EC decision criteria established for quantitative screening methods. For this purpose calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness were evaluated. The proposed method was applied to other AFWS with satisfactory results.     

Method development validation for corticoids in animal feed samples by liquid chromatography using a monolithic column

A LC method for corticosteroids (CC) determination in poultry feed using a Chromolith column and UV detection has been developed and validated. The method development involved the optimization of different hydro-organic mobile phases using methanol or ACN as organic modifiers, flow rate, and temperature. The optimum separation was achieved at 40 degrees C using ACN/water (21:79 v/v) as mobile phase and 3 mL/min flow rate, allowing the separation to baseline of four out of seven CC in about 10 min. Prior to LC, a sample preparation procedure previously assayed for anabolics was used. It includes a leaching process, saponification of the esters from fatty acids, and SPE. Method validation was carried out according to the EU criteria established for quantitative screening methods. The extraction efficiencies, decision limits (CCalpha), and detection capabilities (CCbeta) for these compounds were in the ranges of 86-92%, 27-36 microg/kg, and 33-43 microg/kg, respectively. The repeatability and the within-laboratory reproducibility at 1, 1.5, and 2 CCbeta concentration levels were smaller than 9.0, 5.0, and 4.2% and 9.4, 6.4, and 4.9%, respectively. The CV values of the robustness test were less than 3.8% and the accuracy was in the range of 98-103%. The proposed method was applied to other feed with satisfactory results.     

Liquid chromatographic method development for steroids determination (corticoids and anabolics). Application to animal feed samples

A LC isocratic separation study of a complex mixture containing 18 steroids (corticoids and anabolics), used potentially as growth promoters, was carried out. For this purpose, using a Hypersil ODS column at controlled temperature, mobile phases (from binary to quaternary) prepared from water and MeOH, ACN or THF as organic modifiers and UV detection at 245 nm, were employed (dehydroepiandrosterone was detected at 200 nm). The optimum separation was achieved using water/acetonitrile (65:35, v/v) as mobile phase at 30 degrees C, allowing the separation of 16 out of 18 steroids in about 30 min. The retention scale using optimized binary mobile phases was related with steroids hydrophobicity and structure, allowing a classification into three groups for these compounds. To improve the separation several alkyl-silica packings were tested: Type A (Lichrospher C8) and Type B (Luna C18, Kromasil C18, Purospher C18 and Synergy C12). Taking into account resolution, number of separated compounds and run time analysis the Hypersil column was selected as the best choice for further applications. Calibration graphs were obtained using fluorocortisone, fluoxymesterone or methylprednisolone as internal standard. The optimized separation was applied to the analysis of piglet feed samples spiked with steroids. The sample preparation process included solvent extraction using diethyleter and solid phase extraction using silica cartridges. The recoveries were in the range 70-92%. Decision limits and detection capability were in the range 34-198 and 41-249 microg/kg, respectively. Repeatability was also evaluated.     

Method development for cortisol and cortisone by micellar liquid chromatography using sodium dodecyl sulphate: application to urine samples of rugby players

The chromatographic behavior of cortisol and cortisone using a micellar medium of sodium dodecyl sulphate (SDS) as surfactant, a Hypersil C18 (150- x 3.2-mm i.d., 5 microm) column, a flow rate of 0.5 mL/min, and UV absorbance detection at 245 nm is described. The effect of several organic modifiers and the surfactant concentration on the separation is studied. A mobile phase of 18 mM SDS and 8.3% tetrahydrofuran allows for the separation of cortisol and cortisone up to baseline. These results are also achieved by applying a bivariant optimization method. The proposed method is sensitive, reproducible, and selective. In addition, it is less expensive than conventional high-performance liquid chromatography methods for cortisol and cortisone. The method is applied to the determination of cortisol and cortisone in urine samples of rugby players before and after stress for doping control purposes.     

Comparison of the performance of conventional microparticulates and monolithic reversed-phase columns for liquid chromatography separation of eleven pollutant phenols

The performance of isocratic separations of 11 pollutant phenols (PP) using monolithic (Chromolith RP-18e) and conventional reversed-phase 5 microm (Luna and Purospher C18) and 4 microm (Synergi C12) particulate size columns, selected from high purity silica materials, has been compared. The separations have been optimized based on a previously optimized separation in which a reversed-phase C18 Luna column and acetonitrile as organic modifier were used, allowing the separation of all phenols tested in 23 min. The optimization process was carried out for each column by studying the effect of the mobile phase (acetonitrile as organic modifier, pH, flow-rate) on phenols separation. Under the optimized separation conditions, all phenols were separated in less than 23 min for all columns tested. Asymmetry factors were further evaluated and used to estimate column efficiency using the Dorsey-Foley equation. The efficiency and asymmetry factors were lower for Chromolith than for Purospher and Luna columns respectively. The Chromolith column was finally selected, due to its lower flow resistance, analysis time and good efficiency and asymmetry factors. The PPs separation was achieved in 3 min. The asymmetry factors were in the range 0.9-1.5 using 50mM acetate buffer (pH = 5.25)-ACN (64:36, v/v) as mobile phase, T=45 degrees C and 4.0 ml min(-1) flow-rate.     

Liquid chromatographic method development for anabolic androgenic steroids using a monolithic column Application to animal feed samples

An isocratic HPLC method for the determination with screening purposes of anabolic androgenic steroids (AASs: fluoxymesterone, boldenone, nortestosterone, metandrostenolone, norethindrone, methyltestosterone and bolasterone), used as growth promoting agents, in finishing pig feed samples has been developed and validated. The separation was achieved by using a reversed-phase Chromolith RP-18e column at controlled temperature, UV-detection at 245 nm and epitestosterone as internal standard. The method development involved optimization of different aqueous-organic mobile phases using methanol or acetonitrile as organic modifiers, flow-rate and temperature. The optimum separation for these compounds was achieved at 40 °C using ultrapure water:acetonitrile (71:29, v/v) as mobile phase and 3 mL min⁻¹ flow-rate, allowing the separation of AASs with baseline resolution in about 15 min. The optimized method was applied to the analysis of AASs in finishing pig feed samples. Prior to HPLC, sample preparation procedure was used by leaching using acetonitrile, saponification in a basic medium and solid-phase extraction using polymeric Abselut Nexus cartridges. Method validation has been carried out according to the European Commission Decision 2002/657/EC. The extraction efficiencies, decision limits (CCα) and detection capabilities (CCβ) for these compounds were in the range 83-96%, 27-37 and 32-47 μg kg⁻¹ range, respectively. The within-laboratory reproducibility at 1, 1.5 and 2 CCβ concentration levels were smaller than 13, 10 and 8%, respectively. Finally, the proposed method was successfully applied to nine different kinds of animal feed.     

GC-MS method development and validation for anabolic steroids in feed samples

A GC-MS method for the determination of AAS used as growth promoting agents using SIM in piglet feed samples has been developed and validated, using testosterone as internal standard. The formation of volatile steroid derivatives was carried out by derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide. The optimum separation was achieved using a Zebron ZB-5 column under a gradient temperature elution, allowing the separation of steroids in 18 min. The required sample treatment process was discussed. A leaching using ACN, saponification using a binary NaOH/MgCl2 solution, and LLE using ethyl acetate were finally selected. Method validation has been carried out according to the Commission Decision 2002/657/EC criteria established for quantitative confirmatory methods. The extraction efficiencies, CCalpha and CCbeta for these compounds were in the ranges 78-98%, 10-21 and 18-35 mug/kg, respectively. The repeatability and the within-laboratory reproducibility at 1, 1.5, and 2 CCbeta concentration levels were smaller than 8.2, 7.5, and 5.8% and 12.2, 9.5, and 7.5%, respectively. Accuracy was in the 99-103% range. The robustness was evaluated using the Youden robustness test. The proposed method was applied to the analysis of steroids spiked in different kinds of animal feed samples with satisfactory results.     

Comparison of the performance of different reversed-phase columns for liquid chromatography separation of 11 pollutant phenols

A systematic optimization of the HPLC separation of a mixture containing 11 pollutant phenols (PPs) using a Hypersil ODS (250 mm x 4.6 mm, 5 microm) column and UV-DAD detection has been carried out. The binary mobile phases used were obtained by mixing 50 mM phosphate (pH = 3.0) and methanol, ACN, or THF as organic modifiers. After selecting ACN as an organic modifier, the effects of pH and temperature on PPs separation were studied. A mobile phase of 50 mM acetate (pH = 5.0)-ACN (60:40 v/v) at 50 degrees C allowed the separation of 11 phenols but not to baseline in 17 min. To improve the performance of this separation, the following RP columns were tested: Luna C18 (2), Purospher C18, Synergi C12, Synergi Fusion C18, Gemini C18, Luna Cyano, Lichrospher C8, and Envirosep-PP (polymeric). In all the cases, the performance (analysis time, retention, selectivity, resolution, asymmetry factors, and efficiency) was evaluated. A further reoptimization of the mobile phase was carried out for all the columns by studying the ACN content and pH, with the aim of improving the above-mentioned separations and selecting the most suitable one for PPs analysis.     

A liquid chromatography method using a monolithic column for the determination of corticoids in animal feed and animal feeding water

An HPLC-DAD method for determining corticoids in calf feed and in animal feeding water samples using a monolithic column has been developed and validated. The method optimization included the study of binary mobile phases of water and acetonitrile. The optimum separation was achieved at 40 °C, with acetonitrile:H₂O 29:71 v/v used as mobile phase and a 3 ml/min flow-rate, which resulted in their separation in about 5 min. Two reported sample procedures were applied to feed and for animal feeding water samples prior to HPLC. Method validation was carried out according to the EU criteria established for quantitative screening methods. The results indicate that this method is highly specific, reproducible and accurate. The proposed method was found to be robust and unaffected by small variations in the extraction procedure and in HPLC conditions. The developed method for the determination of corticoids in feed and water samples was also found to be suitable for different kinds of feeds and waters.