Subcritical water extraction of thyreostats from bovine muscle followed by liquid chromatography-tandem mass spectrometry.

Thyreostats can be used fraudulently to promote a rapid increase in weight of breeding animals at low cost. Their severe toxicological effects impose the development of reliable analytical methods to be used in monitoring plans. This work describes an alternative approach to isolate residues of thiouracil, methyl-thiouracil, propyl-thiouracil, phenyl-thiouracil, tapazole and mercaptobenzimidazole from bovine muscle tissue. The developed procedure is based on the following three steps: i) matrix solid-phase dispersion with C18 for the preliminary sample preparation; ii) subcritical water extraction (SWE) at 160°C and 100 bar; iii) clean-up on an Oasis HLB cartridge. The quantitative determination was performed by LC-MS/MS in dual polarity ionization by using internal standardization. The SWE-LC-MS/MS method was validated according to the identification criteria of the Commission decision 2002/657/EC. The relative recoveries ranged from 72 to 97%; within-lab reproducibility was less than 18%. The decision limit and the detection capability of all analytes were below the recommended concentration, set at 10 µg kg-1, but the validation results demonstrated that this method could only be applied for screening of thiouracil and methyl-thiouracil. Besides the analytical advantages related to the use of water as solvent extraction, the procedure allowed significant removal of lipids, whose detrimental effects on instrumentation and MS sensitivity are well-known.

Quantitative profiling of retinyl esters in milk from different ruminant species by using high performance liquid chromatography-diode array detection-tandem mass spectrometry.

An effective high performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) analytical approach was developed for retinoid profiling in raw milk samples (cow, buffalo, ewe, and goat). The analytes were isolated by means of liquid-liquid extraction, including a "lipid freezing" step, with yields exceeding 66%. Since the positive atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) detection is not completely selective, a reliable identification has been accomplished by fully separating the analytes on a tandem C18/C30 column system under non-aqueous reversed phase (NARP) chromatography conditions. After validation, different milk varieties obtained from pasture-fed animals were analysed, providing, for the first time, the retinoid composition of both buffalo's and ewe's milk. According to the literature, retinyl palmitate has been found to be the most abundant vitamin A vitamer, but retinyl oleate is the prevalent form in the caprine milk.

Use of a Novel Sub-2 µm Silica Hydride Vancomycin Stationary Phase in Nano-Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers.

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano-liquid chromatography (nano-LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns-AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74-4.17 and 1.39-1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41-6.09 and α 1.28-2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min.

Enantiomers separation by nano-liquid chromatography: use of a novel sub-2 µm vancomycin silica hydride stationary phase.

A novel sub-2 µm chiral stationary phase (CSP) was prepared immobilizing vancomycin onto 1.8 µm diol hydride-based silica particles. The CSP was packed into fused silica capillaries of 75 µm i.d. with a length of 11 cm and evaluated by means of nano-liquid chromatography (nano-LC) using model compounds of both pharmaceutical and environmental interest (some non-steroidal anti-inflammatory drugs, ß-blockers and herbicides). The study of the effect of the linear velocity of the mobile phase on chromatographic efficiency showed good enantioresolutions up to a value of 5.11 at the optimal linear velocity with efficiencies in terms of number of plates per meter in the range 51,650-68,330. The results were compared with the ones obtained employing 5 µm vancomycin modified diol-silica particles packed in capillaries of the same i.d. For the acidic analytes the sub-2 µm CSP showed better performances, the baseline chiral separation of several studied compounds occurred in an analysis time of less than 3 min. Column-to-column packing reproducibility (n=3) expressed as relative standard deviation was in the range 2.2-5.8% and 0.5-7.7% for retention times and peak areas, respectively.

Effect of content of chiral selector and pore size of core-shell type silica support on the performance of amylose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phases in nano-liquid chromatography and capillary electrochromatography.

In this study the separation performance of various chiral stationary phases (CSPs) made of polysaccharide-based chiral selectors coated onto superficially porous (core-shell or fused-core) silica supports were evaluated. The CSPs obtained by coating of various amounts of chiral selector (1-5%) onto supports of various pore size (100 and 300 Å) were studied. Their evaluation was pursued in both chiral nano-liquid chromatography (nano-LC) and chiral capillary electrochromatography (CEC). Among the goals of this study was to re-examine our previous unexpected finding of better performance of superficially porous CSP under CEC conditions compared to nano-LC conditions for a new set of chiral compounds, as well as to study the effect of varying the chiral selector content and nominal pore size of supporting silica on the performance of core-shell silica-based polysaccharide-type CSPs. Based on the results of this study it can be seen that CSPs based on superficially porous silica can successfully be used for the separation of enantiomers in both nano-LC and CEC mode. Only a slight advantage of CEC over nano-LC mode was observed in this study from the viewpoint of plate numbers, especially at higher mobile phase flow rates. It must also be noted that the optimal theoretical plate height is still too high and further optimization of superficially porous CSPs is necessary for both nano-LC and CEC applications.

Current applications of miniaturized chromatographic and electrophoretic techniques in drug analysis.

In the last decade, miniaturized separation techniques have become greatly popular in pharmaceutical analysis. Miniaturized separation methods are increasingly utilized in all processes of drug discovery as well as quality control of pharmaceutical preparation. The great advantages presented by the analytical miniaturized techniques, including high separation efficiency and resolution, rapid analysis and minimal consumption of reagents and samples, make them an attractive alternative to the conventional chromatographic methods for drug analysis. The purpose of this review is to give a general overview of the applicability of capillary electrophoresis (CE), capillary electrochromatography (CEC) and micro/capillary/nano-liquid chromatography (micro-LC/CLC/nano-LC) for the analysis of pharmaceutical formulations, active pharmaceutical ingredients (API), drug impurity testing, chiral drug separation, determination of drugs and metabolites in biological fluids. The results concerning the use of CEC, micro-LC, CLC, and nano-LC in the period 2009-2013, while for CE, those from 2012 up to the review draft are here summarized and some specific examples are discussed.

Use of novel phenyl-hexyl core-shell particles in nano-LC.

This paper reports the use of novel phenyl-hexyl core-shell particles packed into fused silica capillaries in nano-LC. Capillary columns of different id of 25, 50, 75, 100, and 150 µm were packed employing the slurry packing method. The columns were used for the separation of a model mixture containing five aromatic hydrocarbons. Benzene, toluene, ethylbenzene, n-propylbenzene, and n-butylbenzene were separated utilizing an isocratic elution mode. Mixtures of water/ACN at different ratio were studied to find optimal experimental conditions for baseline separation of all sample components. As expected with this novel stationary phase, an RP chromatographic mechanism was observed. A mixture of water/ACN, 30:70, v/v allowed the complete resolution of the studied analytes. Efficiency increased by decreasing the capillary id recording the highest number of plates per meter with capillaries of 25 µm id. The decrease of the column id also resulted in a flatter dependence of the plate numbers on the linear flow rate of the mobile phase allowing the increase of the flow rate of the mobile phase without significant decrease of efficiency.

Nano-liquid chromatography coupled with mass spectrometry: separation of sulfonamides employing non-porous core-shell particles.

In this paper, nano-liquid chromatography coupled with mass spectrometry was used for the simultaneous determination of 18 sulfonamides. Preliminary experiments were carried out to investigate selectivity of three different stationary phases. Best results were achieved utilizing a capillary column (100 µm I.D.) packed in our laboratory following the slurry packing procedure with a recent commercialized stationary phase, Kinetex(®) C(18) core-shell. A binary mobile phase, consisting of water and acetonitrile and both containing 0.1% (v/v) formic acid, was employed in a gradient mode at a low flow rate (190 nL/min). Applying these conditions, baseline separation of studied compounds was obtained in about 35 min. Although sulfathiazole and sulfapyridine were resolved from the other drugs, they were not separated from each other. Sensitivity was improved by on-column focusing evaluating the appropriate sample solvent and establishing the maximum injection volume. Comparison between UV and mass spectrometric detection was done. Milk samples spiked with sulfonamides were subjected to a rapid pre-treatment by means of acidic deproteinization followed by solid-phase extraction, and analyzed with the developed method. The detection limits obtained in milk were in the range of 8-96 µg/kg, however they were low enough according to the regulations set-up by the European Commission. Calibration curves showed good linearity (R(2)>0.995) in the studied concentration ranges.