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.

Miniaturized extraction methods of triclosan from aqueous and fish roe samples. Bioconcentration studies in zebrafish larvae (Danio rerio).

Triclosan, an antibacterial and antifungal agent, is widely used in household and personal care products, processed foods and food packaging, etc., and thus also released into the environment. Triclosan is acutely and chronically toxic to aquatic organisms and bioaccumulates in fish tissue. Here, we propose a new miniaturized triclosan extraction method for aqueous and fish roe samples, based on the use of a vortex mixer and an ultrasonic probe, respectively, and useful for triclosan determination by gas chromatography coupled to a micro electron capture detector. Different solvents for extraction and sorbents for clean-up purposes were tested. Multivariate optimization of the variables affecting ultrasonic extraction (ultrasound radiation amplitude, sonication time, sample temperature, and the ratio of sample amount and extracting volume) was carried out. Solvent extraction using ethyl acetate and further clean-up with mixed bed cartridges with two layers of Florisil® and Florisil® impregnated with 10% sulfuric acid only for fish roe samples was finally selected. Extraction efficiencies of up to 95% and 90%, and detection limits of 0.165 ng ml(-1) and 2.7 ng g(-1) for aqueous and fish roe samples were obtained, respectively. The optimized method was used in bioconcentration studies with zebrafish larvae (Danio rerio), as an alternative method to the Organization for Economic Cooperation and Development technical guideline 305. Bioconcentration values, BCF = 2,630 and 2,018 at exposure concentrations of 30 and 3 µg L(-1), respectively, were assessed. These results are in agreement with those reported in the literature, showing the feasibility of the method for estimation of toxicokinetic parameters and bioconcentration factors using zebrafish larvae instead of adult fishes, reducing considerable animal testing, as suggested by the European legislation.

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.

New approach to optimize HPLC separations of acid-base compounds with elution order involved, by using combined three-band resolution maps.

Some of the optimization methods in reversed phase high-performance liquid chromatography (RP-HPLC) are based on resolution of the critical band pair. Mobile phase composition is changed systematically to establish those conditions giving an acceptable resolution for such a critical band pair, but sometimes the critical pair may change with the separation conditions, which obliges to identify it for each of those conditions. In the case of ionizable compounds, more than two bands may be involved in resolution, showing--in some cases--changes in the elution order when the mobile phase composition was modified. In this paper, an alternative way that does not identify the critical pair after changing experimental conditions is proposed. The relative separation of the three bands involved in two alternating critical band pairs is evaluated as a sort of conjugate or combined resolution, represented as contour maps vs. two variables (content of organic modifier and pH). These maps are obtained from data of chromatograms made under different separation conditions; these conditions were generated by experimental design and data was mathematically processed with a computer program. Analytes of three families that have acid-base properties, triazines, phenoxyacids, and phenols, were used for this purpose. The chromatographic behavior when elution order reversion of ionizable compounds exists is studied.

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 degrees C, with acetonitrile:H(2)O 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.

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.

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.

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.

HPLC method development for testosterone propionate and cipionate in oil-based injectables.

Two isocratic liquid chromatographic methods for the determination of testosterone propionate (TP) and cipionate (TC) in oil-based injectables using methyltestosterone and bolasterone as internal standards, respectively, have been developed and validated. Mobile phases 57% water:acetonitrile 43% (v:v) and 54% water:acetonitrile 46% (v:v) were used for TP and TC, respectively. For both methods, a bonded-silica Luna CN (250 mm x 4.6 mm i.d., 5 microm) (25 degrees C) column, a flow-rate 1 ml min(-1) and UV absorbance detection at 245 nm were used and two separations up to base line were achieved. Prior to HPLC analysis, sample preparation was required, including extraction of TP and TC from oil-based injectables using the surfactant sodium dodecyl sulphate.

Method development for betamethasone and dexamethasone by micellar liquid chromatography using cetyl trimethyl ammonium bromide and validation in tablets. Application to cocktails.

An isocratic liquid chromatographic method for the determination of betamethasone (BM) and dexamethasone (DM) using methylprednisolone (MPL) as internal standard and micellar mobile phases consisting of cetyl trimethyl ammonium bromide (CTAB) and organic modifiers such as propanol, butanol and pentanol has been developed. The effect of organic modifiers, surfactant concentration, temperature and flow-rate on the separation has been studied. Method validation for dexametasone or bethametasone in tablets was carried out using a mobile phase 0.24% pentanol and 32.5 mM CTAB, a flow-rate of 0.5 ml min(-1), an Hypersil C18 column (60 degrees C), and UV detection at 243 nm. The recoveries for BM and DM found in the accuracy test were 99 +/- 3 and 101 +/- 2, respectively. Repeatability and intermediate precision expressed as R.S.D. were lower than 5% for both compounds. The proposed method was applied to cocktails containing both compounds.

Optimization of the separation of a complex mixture of natural and synthetic anabolic steroids by micellar liquid chromatography.

A systematic optimization of the HPLC separation of a complex mixture containing natural and synthetic anabolic steroids by micellar liquid chromatography using a Hypersil (150 mm x 3.0 mm i.d., 5 microm) C18 column and UV detection at 245 nm (exception is made for oxymetolone and danazol which were monitorized at 280 nm) has been carried out. The isocratic micellar mobile phases (from binary to quaternary) consisted of sodium dodecyl sulphate and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol. The effect of the organic modifiers, surfactant concentration, temperature, ionic strength and flow-rate on the separation has been studied. A micellar mobile phase 5% propanol and 40 mM surfactant allowed the separation of 12 steroids out of 14 tested in about 20 min. A bivariant optimization method for the micellar mobile phase propanol-surfactant corroborated the above results.

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.

Method development for corticosteroids and anabolic steroids by micellar liquid chromatography.

A systematic optimization of the HPLC separation of a complex mixture containing urinary steroids (anabolics and corticoids), boldenone and bolasterone (synthetic anabolics) by micellar liquid chromatography has been carried out. The isocratic micellar mobile phases (from binary to quaternary) consisted of sodium dodecyl sulphate and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol. The effect of the organic modifiers, surfactant concentration, temperature, ionic strength and flow-rate on the separation has been studied. A micellar mobile phase made of 5% propanol and 40 mM surfactant allowed the separation of 13 steroids in about 23 min. A bivariant optimization method for the micellar mobile phase surfactant-propanol corroborated the above results. The separations obtained show good perspectives for future developments.

Development and method validation for testosterone and epitestosterone in human urine samples by liquid chromatography applications.

An isocratic high-performance liquid chromatographic method for the determination of testosterone (T) and epitestosterone (ET) in human urine using liquid-liquid or solid-phase extraction (SPE) is developed and validated. The optimum separation is achieved using a Hypersil C(18) column, water-acetonitrile (57:43, v/v) as the mobile phase and UV-absorbance detection at 245 nm. The recoveries obtained for T and ET in liquid-liquid and SPE demonstrate that these procedures are interchangeable. Quantitation limits for T and ET are 8.6 and 5.4 ng/mL using solvent extraction and 7.3 and 5.7 ng/mL using SPE, respectively. The proposed method is used to evaluate the urinary T, ET, and the T/ET ratio for a healthy male population using liquid-liquid extraction, and the T and ET excretion profile for nine healthy men using SPE.

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.

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.

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.

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.

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 Cushing's syndrome.