A. Santos-Montes

Method development and validation for melamine and its derivatives in rice concentrates by liquid chromatography. Application to animal feed samples

An isocratic LC method for the determination of melamine and its degradation products (ammelide, ammeline, and cyanuric acid), used to increase the apparent protein content of rice protein concentrate, has been developed. Method development involved optimization of different RP columns, aqueous mobile phases, pH, phosphate concentration, and temperature. The optimum separation of these compounds was achieved using a Luna CN column (30xa0°C), 5xa0mmol L−1 sodium phosphate (pH 5.0) as mobile phase, 1xa0mL min−1 flow-rate, UV absorbance-DAD detection at 220xa0nm, and resorcine as internal standard; this enabled separation of these compounds with baseline resolution (values in the 2.1–10.1 range) in about 8xa0min. Prior to HPLC, the developed sample preparation procedure consisted in a leaching process using the above mentioned mobile phase. Method validation was carried out in rice protein concentrates in accordance with the European Commission decision 2002/657/EC criteria. For this purpose, eight mandatory performance characteristics for the conventional validation approach were determined: calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness. The extraction efficiencies for these compounds were in the range 99–100% and the within-laboratory reproducibility at 1.0, 1.5, and 2.0 detection capabilities concentration levels were smaller than 5, 4, and 3%, respectively. Finally, the proposed method was successfully applied to the analysis of other rice protein concentrates and several animal feed samples.

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

SummaryAn HPLC method for the simultaneous determination of the glucocorticoids betamethasone and dexamethasone is described. The method based on the separation of these compounds using a binary watertetrahydrofuran mobile phase and a reversed-phase Hypersil C18 column, was applied to the determination of betamethasone and dexamethasone in tablets.

Optimization of separation of a complex mixture of natural and synthetic corticoids by micellar liquid chromatography using sodium dodecyl sulphate. Application to urine samples.

A systematic optimization of the separation of a mixture of corticoids by micellar liquid chromatography, using sodium dodecyl sulphate as surfactant, a Hypersil (250 mm x 3.2 mm I.D.) C18 column, a flow-rate of 0.5 ml min(-1), and UV absorbance detection at 245 nm has been carried out. Several mobile phases consisting of sodium dodecyl sulphate and different organic modifiers were tested of which tetrahydrofuran, PrOH and BuOH were finally selected. On the basis of analysis time, resolution and number of compounds separated, a mobile phase containing 36 mM sodium dodecyl sulphate and 1.91% butanol allowed the separation of thirteen corticoids out of sixteen in about 27 min. Under these conditions the optimal concentration of sodium dodecyl sulphate was found to be 36 mM. A bivariant optimization method for the mobile phase BuOH-sodium dodecyl sulphate corrobored these results. The effects of temperature, ionic strength and flow-rate effect have also been studied. The most important analytical figures of merit were assessed and compared with those obtained using conventional mobile phases. The optimized method was applied to human urine samples of subjects administered with Dezacor (tablets containing 30 mg of the active ingredient deflazacort) with and without sample preparation.

Simultaneous determination of cortisol and cortisone in urine by reversed-phase high-performance liquid chromatography. Clinical and doping control applications.

A reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of cortisol and cortisone in human urine samples using methylprednisolone as the internal standard is described. The method involves the systematic use of isocratic mobile phases of water and methanol, acetonitrile or tetrahydrofuran and a reversed-phase Hypersil C18 column. A water-acetonitrile mixture used as the mobile phase proved to be the most adequate one for analyzing urine samples purified by solvent extraction. The proposed method is sensitive, reproducible and selective. It was applied to the determination of cortisol and cortisone in several human urine samples: healthy subjects, sportsmen before and/or after stress for doping control purposes, and patients with Cushings syndrome.

High-performance liquid chromatographic separation of a complex mixture of diuretics using a micellar mobile phase of sodium dodecyl sulphate: Application to human urine samples

A systematic optimization of the HPLC separation of a complex mixture containing 19 diuretics by micellar liquid chromatography using sodium dodecyl sulphate (SDS), a Hypersil (150 mmx3.0 mm I.D., 5 microm) C18 column, a flow-rate of 0.5 ml min(-1) and UV absorbance detection has been carried out. Several mobile phases consisting of SDS and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol, and the pH adjusted to 3.2, were tested. The effect of the organic modifier and SDS concentration on the retention behavior and separation of the diuretics was investigated. A mobile phase containing 40 mM SDS and 4% tetrahydrofuran was finally selected. Under these conditions, 14 out of 19 diuretics were separated in about 31 min. A bivariant optimization method for the mobile phase SDS-tetrahydrofuran corroborated the above results. The effect of temperature on the retention was also studied, and 50 degrees C was selected. The optimized method was applied to human urine samples of subjects administered Diurex (tablets containing 20 mg of the active ingredient xipamide) without sample preparation.

Optimization of the high-performance liquid chromatographic separation of a mixture of natural and synthetic corticosteroids

Systematic optimization of the HPLC separation of a mixture of natural and synthetic corticosteroids was carried out for screening purposes. The method involves binary, ternary or quaternary mixtures containing water, methanol, acetonitrile and tetrahydrofuran. It was possible to separate thirteen out of fourteen corticosteroids contained in a sample in about 26 min, with a 5-microns Hypersil-C18 (250 mm x 4.6 mm I.D.) column thermostated at 30 degrees C, using a mobile phase composed of water-tetrahydrofuran (72:28, v/v). This separation was not improved using other C8 or C18 columns. The effect of temperature on the separation of these compounds was also studied. Calibration graphs were established for each corticosteroid up to 8 micrograms/ml using indapamide as internal standard. The detection limits were in the range 0.02-0.14 ng. The optimized method was applied to urine samples spiked with corticosteroids and showed potential for future applications.

Solvent and solid-phase extraction of natural and synthetic corticoids in human urine

Optimization of the main variables that affect solvent and solid-phase extraction processes, using disposable C18 cartridges and the non-ionic polymeric resin Serdolit AD-2, of human urine containing natural and synthetic corticoids is described. The data were obtained from different HPLC separations of these compounds using calibration graphs obtained before and after extraction of these compounds. The procedures, including sample preconcentration, showed efficiencies over 90%. NaCl was used to avoid emulsion formation in solvent extraction. The results achieved using solvent and solid-phase extraction are discussed.

Extraction and high-performance liquid chromatographic separation of deflazacort and its metabolite 21-hydroxydeflazacort application to urine samples

Two HPLC methods for the separation of a mixture of corticoids including the oxazolinic corticoid deflazacort and its metabolite 21-hydroxydeflazacort using different water-tetrahydrofuran mobile phases were developed. Both separations allowed the detection and determination of fifteen out of sixteen corticoids using different C18 columns. Extraction data for deflazacort and its metabolite using different extraction procedures are also reported. These separation conditions were applied to urine samples from two male volunteers administered Dezacor, with both doping control and clinical purposes.

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 40xa0°C, with acetonitrile:H2O 29:71 v/v used as mobile phase and a 3xa0ml/min flow-rate, which resulted in their separation in about 5xa0min. 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.

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 245nm 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 degrees C using ultrapure water:acetonitrile (71:29, v/v) as mobile phase and 3mLmin(-1) flow-rate, allowing the separation of AASs with baseline resolution in about 15min. 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 (CCalpha) and detection capabilities (CCbeta) for these compounds were in the range 83-96%, 27-37 and 32-47microgkg(-1) range, respectively. The within-laboratory reproducibility at 1, 1.5 and 2 CCbeta concentration levels were smaller than 13, 10 and 8%, respectively. Finally, the proposed method was successfully applied to nine different kinds of animal feed.