Antonio L. Crego

Recent advances in the analysis of antibiotics by CE and CEC.

This article reviews the recent developments achieved in the analysis of antibiotics by CE and CEC. CZE has mainly been employed although other separation modes have also been used. Different detection systems coupled to CE have been reported for antibiotic determination. Enhancement on sensitivity through the use of diverse offline, inline, and online preconcentration approaches is also described. Special attention has been paid to sample treatment steps and to the applications developed in different research areas such as the pharmaceutical, biological, food, and environmental. This manuscript covers the period of time from the previous review published in June 2009 until May 2011.

About the role of enantioselective selector-selectand interactions and the mobilities of diastereomeric associates in enantiomer separations using CE

It is generally accepted that the selective binding of enantiomers of the chiral analyte to a chiral selector is necessary for enantioseparations in CE, whereas the role of mobility differences between the temporary diastereomeric associates formed between the enantiomers and the chiral selector has been commonly neglected. One of the authors of this study suggested in 1997 that the mobility difference between the diastereomeric associates of two enantiomers with the chiral selector may be solely responsible for a separation of enantiomers in CE and enantioselective selector–selectand binding may be not necessarily required. Several indirect confirmations of this hypothesis have been described in the literature within the last few years but a dedicated study proving this concept has not been published yet. The present data obtained for the two chiral antimycotic drugs ketoconazole and terconazole by CE and NMR spectroscopy unequivocally support this concept.

Recent approaches in sensitive enantioseparations by CE.

The latest strategies and instrumental improvements for enhancing the detection sensitivity in chiral analysis by CE are reviewed in this work. Following the previous reviews by García‐Ruiz et al. (Electrophoresis 2006, 27, 195–212) and Sánchez‐Hernández et al. (Electrophoresis 2008, 29, 237–251; Electrophoresis 2010, 31, 28–43), this review includes those papers that were published during the period from June 2009 to May 2011. These works describe the use of offline and online sample treatment techniques, online sample preconcentration techniques based on electrophoretic principles, and alternative detection systems to UV–Vis to increase the detection sensitivity. The application of the above‐mentioned strategies, either alone or combined, to improve the sensitivity in the enantiomeric analysis of a broad range of samples, such as pharmaceutical, biological, food and environmental samples, enables to decrease the limits of detection up to 10−12 M. The use of microchips to achieve sensitive chiral separations is also discussed.

Sensitive determination of d-carnitine as enantiomeric impurity of levo-carnitine in pharmaceutical formulations by capillary electrophoresis–tandem mass spectrometry

In this work, capillary electrophoresis-electrospray ionization-tandem mass spectrometry was applied to the determination of l- and d-carnitine in pharmaceutical formulations. A simple sample treatment procedure consisting of the use of a dilution or an extraction step with water was employed prior to derivatization with 9-fluorenylmethoxycarbonyl (FMOC). The method was validated in terms of selectivity, linearity, accuracy, precision and sensitivity, with a LOD of 10ngmL(-1) for each enantiomer, which was enough to detect enantiomeric impurities up to 0.002% of d-carnitine with respect to the main enantiomer (l-carnitine). Eleven pharmaceutical formulations were analyzed including ampoules, oral solutions, sachets, and tablets. Results showed contents for carnitine comprised between 77 and 101% with respect to the labeled ones in the case of those formulations marketed with the racemate, and from 97 to 102% in those cases where the single enantiomer (l-carnitine) was employed as active ingredient. Percentages for the enantiomeric impurity (d-carnitine) ranging from 0.6 to 1.3% were obtained exceeding the limits established for impurities in drug products. These results corroborate the need of validated analytical methodologies enabling the quality control of pharmaceutical formulations containing carnitine.

Development of a CE-MS2 method for the enantiomeric separation of L/D-carnitine: Application to the analysis of infant formulas

A new chiral analytical method based on CE‐MS is proposed for the identification and simultaneous quantification of D/L‐carnitine in infant formulas. Previous derivatization of carnitine with FMOC enabled the optimization of the chiral separation using CE with UV detection. An optimization of electrospray‐MS parameters using a partial filling of the non‐volatile chiral selector (succinyl‐γ‐CD) was performed. A selective fragmentation using MS2 experiments with an ion trap analyser was carried out to confirm the identity of D/L‐carnitine according to the current legislation. Satisfactory results were obtained in terms of linearity, precision, and accuracy. Interestingly, the CE‐MS2 method developed allowed a sensitivity enhancement with respect to UV detection of 100‐fold, obtaining an LOD of 100 ng/g for D‐carnitine. The determination of L‐carnitine and its enantiomeric purity in 14 infant formulas supplemented with carnitine was successfully achieved, sample preparation only requiring an ultrafiltration with centrifugal filter devices to retain the components with the highest molecular weights.

Influence of mobile phase composition on electroosmotic flow velocity, solute retention and column efficiency in open-tubular reversed-phase capillary electrochromatography.

The effects of some experimental parameters, such as the volume fraction and type of organic modifier in the mobile phase, and the concentration, type and pH of the buffer on the electroosmotic flow velocity, the retention behavior of test solutes, and the column efficiency have been investigated in capillary electrochromatography (CEC) using an open-tubular column of 9.60 microm I.D. with a porous silica layer chemically modified with C18 as stationary phase. The retention of a group of polycyclic aromatic hydrocarbons (PAHs) used as a test mixture varied significantly by changing the organic modifier content in the hydroorganic mobile phase according to the reversed-phase-like selectivity of the stationary phase. In addition, an increase in the percentage of organic modifier resulted in a slight increase in the linear velocity of the EOF. On the other hand, when the phosphate buffer concentration was increased over the range 1-50 mM, the electroosmotic mobility fell dramatically, the retention of the solutes decreased steadily, and the plate height showed a significant increase. The results obtained with phosphate, trishydroxymethylaminomethane or 2-morpholinoethanesulfonic acid as buffers were similar when pH remained constant. Optimization in CEC was essential to achieve further enhancement of separation performance, because the analysis time and separation resolution are essentially affected when varying operating parameters. Separations of seven PAHs with more than 100000 plates are presented within 4 min analysis time.

Separation of enantiomers of norephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: Comparative CE and NMR studies

In this study, the enantiomer migration order (EMO) of norephedrine (NEP) in the presence of various CDs was investigated by CE. NMR and CE techniques were used to analyze the mechanism of the chiral recognition between NEP enantiomers and four CDs, i.e., native α‐CD, β‐CD, heptakis(2,3‐di‐O‐acetyl‐6‐O‐sulfo)‐β‐CD (HDAS‐β‐CD), and heptakis(2,3‐di‐O‐methyl‐6‐O‐sulfo)‐β‐CD (HDMS‐β‐CD). EMO was reversed in the presence of α‐CD and β‐CD, although only minor differences in the structures of the complexes formed between NEP and these CDs could be derived from rotating frame nuclear Overhauser experiments (ROESY). The complexes between the enantiomers of NEP and the sulfated CDs, HDMS‐β‐CD, and HDAS‐β‐CD, were substantially different. However, EMO of NEP was identical in the presence of these CDs. HDAS‐β‐CD proved to be the most suitable chiral selector for the CE enantioseparation of NEP.

Enantiomeric separation of FMOC‐amino acids by nano‐LC and CEC using a new chiral stationary phase, cellulose tris(3‐chloro‐4‐methylphenylcarbamate)

A novel polysaccharide‐based chiral stationary phase (CSP), cellulose tris(3‐chloro‐4‐methylphenylcarbamate), also known as Sepapak‐2 or Lux Cellulose‐2, has been evaluated for the enantiomeric separation of FMOC derivatives of amino acids. After mobile‐phase optimization in nano liquid chromatography (nano‐LC) the column enabled the enantiomeric separation of 19 out of 23 amino acids tested, indicating the high chiral recognition power of this new CSP. Subsequently, a comparison of the driving force employed (pressure or voltage) was carried out comparing nano‐LC and CEC under the same conditions. Better peak efficiencies and resolution were observed by using CEC experiments, which enabled the chiral discrimination of 20 out of 23 amino acids tested. Finally, in order to show the potential of this new CSP, the determination of the content and the enantiomeric purity of the non‐protein amino acid citrulline in food supplements was performed. For that purpose, the method was optimized, evaluated and applied to different commercial samples.

Evaluation of new cellulose-based chiral stationary phases Sepapak-2 and Sepapak-4 for the enantiomeric separation of pesticides by nano liquid chromatography and capillary electrochromatography.

Two novel polysaccharide-based chiral stationary phases (CSPs), known as Sepapak-2 (cellulose tris(3-chloro-4-methylphenylcarbamate)) and Sepapak-4 (cellulose tris(4-chloro-3-methylphenylcarbamate)), have been evaluated in this work for the chiral separation of a group of 16 pesticides including herbicides, insecticides and fungicides. The optimization of the mobile phase employed in nano-liquid chromatography (nano-LC) enabled the chiral separation of seven pesticides on Sepapak-2 and of nine pesticides on Sepapak-4. Due to the fact that Sepapak-4 gave better results, this column was selected to compare nano-LC and capillary electrochromatography (CEC) under the same conditions that consisted in the use of a 90/9/1 (v/v/v) ACN/H₂O/ammonium formate (pH 2.5) background electrolyte (BGE). As expected, both the efficiency and the chiral resolution obtained in CEC experiments were higher than in nano-LC for all the analyzed compounds. The analytical characteristics of the CEC developed methodology were evaluated in terms of linearity, LODs, LOQs, precision, selectivity, and accuracy allowing its application to the quantitation of metalaxyl and its enantiomeric impurity in a commercial fungicide product marketed as enantiomerically pure (metalaxyl-M) and in soil and tap water samples after solid phase extraction (SPE). The determined amount of metalaxyl-M was found to be a 26% above the labeled content and it contained an enantiomeric impurity of a 3.7% of S-metalaxyl was determined.

Development of an in-capillary derivatization method by CE for the determination of chiral amino acids in dietary supplements and wines.

A fast in‐capillary derivatization method by CE with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate was developed for the first time for the determination of amino acid enantiomers (arginine, lysine, and ornithine) in dietary supplements and wines. Because of the initial current problems due to the formation of precipitates into the capillary during the derivatization reaction, a washing step with an organic solvent as DMSO between injections was necessary. Different approaches were also investigated to enhance the sensitivity of detection. A derivatization procedure, where plugs of ACN, derivatizing agent (10 mM 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate), and sample in borate (1:1 v/v) were injected in tandem (2, 3, and 6 s, respectively, at 50 mbar), was selected because it enabled to obtain the most sensitive and reproducible results. Appropriate analytical characteristics (linearity, LOD and LOQ, precision, absence of matrix interferences, and accuracy) were obtained for this method. Finally, the optimized method was successfully applied to the determination of the enantiomers of arginine, lysine, and ornithine in food samples of different complexities (dietary supplements and wines).