Maykel Hernández-Mesa

Novel solid phase extraction method for the analysis of 5-nitroimidazoles and metabolites in milk samples by capillary electrophoresis

A new sample treatment has been developed for the extraction of 5-nitroimidazole (5-NDZ) drugs in milk samples previous to their determination by micellar electrokinetic chromatography (MEKC). Fat removing and protein precipitation were simultaneously carried out by the addition of trichloroacetic acid (TCA) and subsequent centrifugation. Clean-up and off-line concentration were achieved by a novel solid-phase extraction (SPE) method employing mixed cation exchange (MCX) cartridges, obtaining an off-line concentration factor of 18. Analyses were performed in less than 18 min employing 20mM phosphate buffer (pH 6.5) and 150 mM SDS as background electrolyte (BGE). During the separation procedure a temperature of 20 °C and a voltage of 25 kV (normal mode) were applied. Due to sweeping effects, an on-line concentration was achieved for all the studied compounds and detection limits lower than 1.8 μg L(-1) were obtained. This method has been successfully applied to milk samples of different origins, including raw ewe milk.

Novel cation selective exhaustive injection-sweeping procedure for 5-nitroimidazole determination in waters by micellar electrokinetic chromatography using dispersive liquid–liquid microextraction☆

A novel method consisting of cation-selective exhaustive injection and sweeping (CSEI-sweeping) as on-line preconcentration followed by a micellar electrokinetic chromatography (MEKC) separation has been developed for the determination of 5-nitroimidazoles (5-NDZ) in environmental waters. Moreover, dispersive liquid-liquid microextraction (DLLME) has been proposed for first time as sample treatment technique prior to CSEI-sweeping-MEKC. DLLME was applied to 5mL of sample. Dibromomethane (1156μL) and 2-butanol (1363μL) were employed as extractant and dispersive solvents, respectively. Salting-out effect was achieved by the addition of 16% (w/v) NaCl to the samples. After DLLME and organic solvent evaporation, the residue was redissolved in a low conductivity solvent (5mM phosphoric acid with 5% of methanol) and electrokinetically injected at 9.8kV for 632s in a bare fused-silica capillary (57.2cm, 50μm I.D.). Prior to the injection, the capillary was rinsed with 50mM phosphate buffer pH 2.5, followed by a plug of a higher conductivity buffer (100mM phosphate pH 2.5, 50mbar, 264s) and a plug of water (50mbar, 2s). Separation was carried out applying -30kV at 20°C in 44mM phosphate buffer pH 2.5, containing 8% tetrahydrofuran and 123mM sodium dodecyl sulfate. Analytical signals were monitored at 276nm. Validation was performed in river and well waters, obtaining satisfactory results in terms of linearity, precision (% RSD generally lower than 10%) and trueness (recoveries higher than 70% in almost all cases). LODs ranged from 0.61 to 2.44ng/mL. The combination of this microextraction technique with the proposed capillary electrophoresis methodology supposes a simple, sensitive and cheap alternative for 5-NDZ analyses, in accordance with the aims of green chemistry.

Green methodology based on dispersive liquid–liquid microextraction and micellar electrokinetic chromatography for 5‐nitroimidazole analysis in water samples

Dispersive liquid-liquid microextraction has been proposed as an extraction technique combined with micellar electrokinetic chromatography (MEKC) for the analysis of eight 5-nitroimidazole compounds, including some metabolites, in water samples. Determination has been carried out using a diode array detector, employing 20 mM sodium phosphate and 150 mM SDS as separation buffer. Separation has taken place under a voltage of 25 kV and a temperature of 20°C. Samples were prepared in a buffer without micelles and they were hydrodynamically injected at 50 mbar for 25 s, producing a sweeping effect on the analytes for increasing sensitivity. Different factors involved in the dispersive liquid-liquid microextraction procedure were optimized, such as sample pH, nature, and volume of extraction and dispersive solvents in the mixture, percentage of NaCl added to sample and shaking time after the injection of the extraction and dispersive solvents. The method was characterized for water samples, achieving detection limits lower than 2.4 μg/L. Trueness was checked in river, tap, and bottled water. Dispersive liquid-liquid microextraction combined with MEKC constitutes an easy, cheap, and green alternative for 5-nitroimidazole analysis in environmental water samples.

Determination of 5-nitroimidazole residues in milk by capillary electrochromatography with packed C18 silica beds.

This work presents a novel methodology for analysing 5-nitroimidazole residues in milk samples by capillary electrochromatography using lab-made packed columns, produced by carrying out a high pressure packing procedure using acetone as driving solvent and C18 silica uncapped particles (5 µm particle size) as packing material. Column frits resulted from sintering the proper stationary phase by heating the packed material for 20s with a nichrome ribbon (80% Ni-20% Cr, 28 cm × 2 mm × 0.2 mm, electric resistance 1.3 Ω) connected to a 7 V AC power supply. Lab-made C18 silica packed capillaries (40 cm x 50 µm i.d.) were employed for the determination of 5-nitroimidazole drugs. Milk samples were treated by a salting-out assisted liquid-liquid extraction followed by a solid phase extraction with Oasis®HLB cartridges prior to their injection. Samples were hydrodynamically injected into the column for 120 s at 11.5 bar. Afterwards eight 5-nitroimidazole compounds were separated in isocratic mode under an applied voltage of 27 kV and a temperature of 30 °C. The selected mobile phase consisted of a mixture 60:40 acetonitrile:ammonium acetate (2.5 mM, pH=5). Separation was monitored at 320 nm and it was performed in less than 15 min. The method was characterized in terms of linearity (R(2)≥0.993) and precision (repeatability, RSD≤12.2% and reproducibility, RSD≤14.5%), obtaining detection limits lower than 29 µg/L for all compounds under study.

Capillary electrochromatography coupled with dispersive liquid-liquid microextraction for the analysis of benzimidazole residues in water samples.

A novel method for the analysis of benzimidazole residues in water samples by capillary electrochromatography-UV detection (290nm), using laboratory-made packed columns is presented. Capillaries (25cm packed length×75µm inner diameter, 34cm total length, 25.5cm effective capillary length) were packed with C18 particles (5µm, non-encapped) following a high pressure packing procedure and using a compact steel unit designed for packing capillary columns. Acetone was employed as solvent to carry the particles through the capillary and pack it under a pressure of 42MPa. Outlet and inlet frits were made by sintering the particles of the stationary phase by heating the packed material with a nichrome ribbon connected to a 7V AC power supply. With the aim of achieving a good analytical performance, the variables that affected the separation were studied, using a mobile phase composition of 60:40 (v/v) acetonitrile/water containing ammonium acetate (1mM, pH 6.5), a separation voltage of 25kV and a temperature of 25°C. In addition, a combined hydrodynamic-electrokinetic injection mode was considered and samples were injected for 75s under a voltage of 12.5kV and a pressure of 11.5bar. Finally, the determination of benzimidazoles in water samples was accomplished by capillary electrochromatography using dispersive liquid-liquid microextraction as sample treatment. Variables affecting the extraction efficiency were optimized, using chloroform and ethanol as extraction and disperser solvents, respectively. This method was applied to different water samples, obtaining satisfactory results in terms of linearity (R2≥0.990), repeatability (RSD≤1.2%), reproducibility (RSD≤2.2%) and trueness (R≥87.7%). Detection and quantification limits were lower than 2.8µgL-1 and 9.3µgL-1, respectively.

Capillary electrochromatography-mass spectrometry for the determination of 5-nitroimidazole antibiotics in urine samples.

The separation of eight antibiotics belonging to 5‐nitroimidazole family was carried out by means of CEC coupled with MS. Preliminary experiments were carried out with ultraviolet detection in order to select the proper stationary and mobile phase. Among the different stationary phases studied (namely Lichrospher C18, 5 μm particle size; CogentTM Bidentate C18, 4.2 μm; Pinnacle II™ Phenyl, 3 μm; Pinnacle II™ Cyano, 3 μm), Cogent™ Bidentate C18 (4.2 μm) gave the best performance. For CEC‐MS coupling, a laboratory assembled liquid‐junction‐nano‐spray interface was used. In order to achieve a good sensitivity, special attention was paid to both optimization of the sheath liquid composition as well as selection of the injection mode. Under optimized CEC‐ESI‐MS conditions, the separation was accomplished within 22 min by using a column packed with a mixture of Bidentate C18:Lichrospher Silica‐60 (5 μm) 3:1 w/w, an inlet pressure of 11 bar, a voltage of 15 kV, and a mobile phase composed by 45:10:45 v/v/v ACN/MeOH/water containing ammonium acetate (5 mM pH 5). A combined hydrodynamic and electrokinetic injection of 8 bar, 15 kV, and 96 s was adopted. The method was validated in terms of repeatability and intermediate precision of retention times and peak areas, linearity, and LODs and LOQs. RSDs values were <2.9% for retention times and <16.1% for peak areas in both intraday and interday experiments. LOQ values were between 0.09 and 0.42 μg/mL for all compounds. Finally, the method was applied to the determination of three most employed 5‐nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in spiked urine samples, subjected to a SPE procedure. Recovery values in the 67–103% range were obtained. Furthermore, for the selected antibiotics, CEC‐MS2 spectra were obtained providing the unambiguous confirmation of these drugs in urine samples.

Development of an ultrasensitive stacking technique for 5-nitroimidazole determination in untreated biological fluids by micellar electrokinetic chromatography

Cation‐selective exhaustive injection and sweeping followed by a MEKC separation is evaluated for the sensitive analysis of 5‐nitroimidazoles in untreated human serum and urine. Deproteinized serum and urine samples were diluted 76 and 143 times, respectively, in a low‐conductivity solvent (5.00 mM orthophosphoric acid containing 5.0% v/v methanol). Samples were electrokinetically injected at 9.8 kV for 632 s in a previously conditioned fused‐silica capillary (65.0 cm × 50 μm id). Separation was performed at –30 kV and 20°C using 44 mM phosphate buffer (pH 2.5), 123 mM SDS, and 8% v/v tetrahydrofurane as BGE. Signals were monitored at 276 nm and peak area was selected as analytical response. Good linearity (R2 ≥ 0.988) and LODs lower than 1.5 and 1.8 μg/mL were achieved in serum and urine, respectively.

Collision Cross Section (CCS) Database: An Additional Measure to Characterize Steroids

Ion mobility spectrometry enhances the performance characteristics of liquid chromatography-mass spectrometry workflows intended to steroid profiling by providing a new separation dimension and a novel characterization parameter, the so-called collision cross section (CCS). This work proposes the first CCS database for 300 steroids (i.e., endogenous, including phase I and phase II metabolites, and exogenous synthetic compounds), which involves 1080 ions and covers the CCS of 127 androgens, 84 estrogens, 50 corticosteroids, and 39 progestagens. This large database provides information related to all the ionized species identified for each steroid in positive electrospray ionization mode as well as for estrogens in negative ionization mode. CCS values have been measured using nitrogen as drift gas in the ion mobility cell. Generally, direct correlation exists between mass-to-charge ratio ( m/ z) and CCS because both are related parameters. However, several steroids mainly steroid glucuronides and steroid esters have been characterized as more compact or elongated molecules than expected. In such cases, CCS results in additional relevant information to retention time and mass spectral data for the identification of steroids. Moreover, several isomeric steroid pairs (e.g., 5β-androstane-3,17-dione and 5α-androstane-3,17-dione) have been separated based on their CCS differences. These results indicate that adding the CCS to databases in analytical workflows increases selectivity, thus improving the confidence in steroids analysis. Consequences in terms of identification and quantification are discussed. Quality criteria and a construction of an interlaboratory reproducibility approach are also reported for the obtained CCS values. The CCS database described here is made publicly available.

Capillary electrophoresis-tandem mass spectrometry combined with molecularly imprinted solid phase extraction as useful tool for the monitoring of 5-nitroimidazoles and their metabolites in urine samples

A novel capillary electrophoresis-tandem mass spectrometry approach is proposed for the determination of eleven 5-nitroimidazoles in urine samples for therapeutical drug monitoring purposes. A comparison between two separation modes, namely micellar electrokinetic chromatography and capillary zone electrophoresis was carried out, obtaining higher selectivity when 1M formic acid (pH 1.8) was selected as background electrolyte. 5-Nitroimidazoles were hydrodynamically injected in water for 40s at 50mbar and their separation was performed at 28kV and 25°C. To improve migration time repeatability, a pressure of 50mbar was applied to the inlet vial during runs without any loss of peak resolution. Electrospray ionization parameters were established as follow: 6L/min, dry gas flow rate; 51,021.2Pa, nebulization pressure; 160°C, dry gas temperature. Sheath liquid consisted of a mixture of propan-2-ol/water/acetic acid (60.0:38.8:0.2% v/v/v) supplied at 3.3µL/min. MS parameters were optimized for analyte identification through their MS2 and MS3 spectra. The method was applied to the determination of 5-nitroimidazoles in urine samples, applying molecularly imprinted solid phase extraction for sample clean-up. Recoveries higher than 79.2% demonstrated the suitability of the procedure. Limits of detection ranged from 9.6 to 130.2µg/L while precision assays resulted in relative standard deviations for peak areas lower than 16.1%.

Evaluation of the combination of micellar electrokinetic capillary chromatography with sweeping and cation selective exhaustive injection for the determination of 5-nitroimidazoles in egg samples.

A methodology is presented for the sensitive determination of nitromidazole residues in egg by means of micellar electrokinetic capillary chromatography in combination with cation selective exhaustive injection and ultraviolet detection. Six compounds have been considered and the separation has been achieved in less than 12min in a 61.5-cm effective length capillary with 50-μm internal diameter. Phosphate buffer 44mM pH 2.5, containing 8% tetrahydrofurane and 123mM sodium dodecyl sulfate was employed as running buffer. Solid phase extraction has been employed for sample clean-up. The methodology has been successfully validated in hen eggs, obtaining method detection limits in the range of 2.1-5.0ng/g. Precision was studied in terms of repeatability and intermediate precision, with relative standard deviations lower than 18.0%. Recoveries were calculated in quail eggs and a commercial pasteurized egg white product, reaching over 70% for most of the considered 5-nitroimidazoles.