R. Gonzalo-Lumbreras

Optimization of the high-performance liquid chromatographic separation of a complex mixture containing urinary steroids, boldenone and bolasterone: application to urine samples

An HPLC separation of a complex mixture containing 13 urinary anabolics and corticoids, and boldenone and bolasterone (synthetic anabolics) has been carried out. The applied optimization method involved the use of binary, ternary and quaternary mobile phases containing acetonitrile, methanol or tetrahydrofuran as organic modifiers. The effect of different reversed-phase packings and temperature on the separation was studied. The optimum separation was achieved by using a water-acetonitrile (60:40, v/v) mobile phase in reversed-phase HPLC at 30 degrees C, allowing the separation of all the analytes in about 24 min. Calibration graphs were obtained using bolasterone or methyltestosterone as internal standards. Detection limits were in the range 0.012-0.107 microg ml(-1). The optimized separation was applied to the analysis, after liquid-liquid extraction, of human urine samples spiked with steroids.

High-performance liquid chromatographic optimization study for the separation of natural and synthetic anabolic steroids. Application to urine and pharmaceutical samples

An HPLC separation of a complex mixture containing 14 androgenic anabolic steroids (natural and synthetic) for screening purposes has been carried out. The applied optimization method involved the use of binary, ternary and quaternary mobile phases containing acetonitrile, methanol or tetrahydrofuran as organic modifiers. The effect of different reversed-phase packings and temperature on the separation using acetonitrile as organic modifier was studied. The optimum separation was achieved by using a water-acetonitrile (55:45, v:v) mobile phase and a Hypersil ODS (250 mm x 4.6 mm) 5 microm column (30 degrees C) in about 38 min, allowing the separation of 14 out of 14 compounds tested (when danazol is excluded, 13 out of 14 were separated in 23 min). Calibration graphs were obtained using bolasterone, methyltestosterone and canrenone as internal standards. Detection limits were in the range 0.012-0.11 microg ml(-1). The optimized separation was applied for monitoring the norethindrone acetate hydrolysis from tablets and to the analysis, after liquid-liquid extraction, of urine samples spiked with steroids.

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.

Solvent and solid-phase extraction of natural and synthetic anabolic steroids in human urine.

Liquid-liquid (using dichloromethane) and liquid-solid extraction processes (using disposable C18 cartridges) were applied to human urine samples spiked with 15 androgenic anabolic steroids (natural and synthetic). The extraction recoveries were assessed from different HPLC separations of anabolic steroids using water-acetonitrile mobile phase, and using calibration graphs obtained by injection into HPLC of standard samples of these compounds before and after extraction. The procedures, including sample preconcentration, showed extraction efficiencies over 90% which were independent on a wide range of concentrations tested. Solid phase extraction yielded poor results for oximetolone, danazol and dehydroepiandrosterone. For real urine samples, hydrolysis using beta-glucuronidase and washing using sodium hydroxide before and after solvent extraction, respectively, is recommended.

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.

Analytical performance of two miniaturised extraction methods for triclosan and methyltriclosan, in fish roe and surimi samples

A new and reliable miniaturised QuEChERS-based extraction method combined with a dispersive SPE cleanup procedure for extracting triclosan and methyltriclosan from fish roe and surimi samples was proposed. The effectiveness of different extraction/partition conditions for QuEChERS method was systematically investigated, and the use of acetonitrile extraction solvent and MgSO4, PSA, C18 and Florisil as cleanup reagents was recommended in the final method. Other method based on ultrasonic extraction with ethylacetate and clean-up with SPE was also evaluated for these samples. Different polymeric and silica sorbents for clean up were tested and the combination of Florisil and PSA was finally selected. The performance of these miniaturised sample preparation methods combined with GC-MS with quadrupole detection were compared. Extraction efficiency as well as cleaning effectiveness, laboriousness and speed were taken as criteria for method evaluation. Satisfactory validation parameters, such as linearity, recovery, precision and LODs and LOQs for both developed analytical methods were obtained from fish roe and surimi samples. Finally, both methods were applied to real samples. The sensitivity of the proposed methods was good enough to ensure reliable determination of target analytes at concentration levels commonly found in this kind of samples.

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.

Capillary liquid chromatography with diode array and mass spectrometry detection for heterocyclic aromatic amine determination in ready-to-eat food treated with electron-beam irradiation

In the present paper, we have developed a capillary liquid chromatography with MS detection for the determination at ngg⁻¹ levels of four heterocyclic aromatic amines (MeIQx, norharman, harman and harmine), a group of mutagenic and carcinogenic compounds that can potentially be produced in protein-rich food during processing operations. They have been determined in commercial ready-to-eat (RTE) smoked salmon and soft cheese treated with E-beam irradiation. On the basis of experimental design studies and operating conditions of MS detector, best chromatographic conditions were obtained using a Luna® C¹⁸ capillary column (150 mm × 0.3 mm I.D.) with a mixture of acetonitrile-ammonium formate 5 mM pH 3.6 buffer (13:87, v/v) as mobile phase. To improve sensitivity, large injection volumes (20 μL) and injection solutions of low elution strength were employed. Sample preparation procedure included a previous treatment with 1M NaOH, followed by two solid-phase extraction steps; firstly on diatomaceous earth and then on mixed-mode cartridges. Heterocyclic amines were detected neither in irradiated and in non-irradiated samples, indicating that they were not formed by the radiation effect even at doses higher than those indicated in the Food Safety Objective established by regulatory agencies. RTE food samples were spiked at concentration levels in the range 10-30 ngg⁻¹. Recoveries higher than 85% (n=3 for each spiked level) were obtained, showing the effectiveness of the proposed methodology.

Optimization and validation of conventional and micellar LC methods for the analysis of methyltestosterone in sugar-coated pills

Two isocratic liquid chromatographic methods (conventional and micellar) for the determination of methyltestosterone in sugar-coated pills using fluoxymesterone as internal standard have been developed and validated. In conventional liquid chromatography a mobile phase 45% water:acetonitrile 55% (v:v), a flow-rate 1 mlmin(-1) and a C(18) Hypersil ODS (250 x 4.6 mm, 5 microm) column (25 degrees C) were used. In micellar liquid chromatography the conditions were: mobile phase 40 mM sodium dodecyl sulfate: 10% propanol, flow-rate 0.5 mlmin(-1) and C(18) Hypersil ODS (150 x 3.0 mm, 5 microm) column (60 degrees C). For both methods, UV absorbance detection at 245 nm was used and a separation up to base line was achieved. Prior to HPLC analysis a simple sample preparation was required.

Conventional and micellar liquid chromatography method development for danazol and validation in capsules

Two isocratic liquid chromatographic methods (conventional and micellar) for the determination of danazol (DZ) in capsules using canrenone (CAN) as internal standard have been developed and validated. In conventional liquid chromatography a mobile phase 35% water:acetonitrile 65%, v:v, a flow-rate 1 ml min(-1) and a C18 Hypersil ODS (250 x 4.6 mm, 5 microm) column (25 degrees C) were used. In micellar liquid chromatography (MLC) the conditions were: mobile phase 40 mM sodium dodecyl sulfate:2% pentanol, flow-rate 0.5 ml min(-1) and C18 Hypersil ODS (150 x 3.0 mm, 5 microm) column (60 degrees C). For both methods. UV absorbance detection at 280 nm was used and a separation up to base line was achieved. Prior to HPLC analysis a simple sample preparation was required. The recoveries found in the accuracy test were 99 +/- 10 and 101 +/- 8%, in conventional liquid chromatography (CLC) and MLC, respectively. Repeatability and intermediate precision expressed as R.S.D. were lower than 5% for both methods. Detection limits obtained were 2.4 and 3.0 ng g(-1) in CLC and CLM, respectively.