Determination of teicoplanin in human plasma by reverse micelle mediated dispersive liquid-liquid microextraction with high performance liquid chromatography.

A reverse micelle mediated dispersive liquid-liquid microextraction (RM-DLLME) combined with high performance liquid chromatography-ultraviolet detector (HPLC-UV) was developed for extraction and determination of 5 A2 components of teicoplanin (TA2-1, TA2-2, TA2-3, TA2-4, TA2-5) in human plasma, and the mechanism of RM-DLLME was analysed and explored. In this method, 80 µL of the reverse micelle solution of cetylpyridinium chloride/n-hexanol (15 mmol/L) was used as the extraction solvent for the separation, extraction and enrichment of the teicoplanin in plasma sample. All factors affecting the extraction efficiencies of the target analytes, such as the amounts of acetonitrile and chloroform, the type and volume of reverse micelle solution, pH and volume of sample phase, dispersant, salt addition, extraction mode and time, centrifugation rate and time, were investigated and optimized. Under the optimum conditions, the 5 A2 components of teicoplanin achieved effective enrichment with the enrichment factors of 228-347 and obtained good linearity in the range of 0.8375-100.5 µg/mL with correlation coefficients higher than 0.9960. The limits of detection were ranged between 0.5025-3.015 µg/mL. Relative standard deviation values of the method precisions were lower than 10.6% and the average recoveries were in the range of 82.7-111.3%. The determination results of the method were demonstrated with favorable characteristics, such as high enrichment, good selectivity and sensitivity, satisfactory precision and accuracy, and this method could be employed to analysis of the teicoplanin in human plasma samples.

Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography.

The objective of this study is to understand the behavior of a peptide in a medium containing supercritical carbon dioxide mixed with an alcohol (methanol) and acidic or basic additives in uncommonly high concentrations. Chirobiotic TAG is a chromatographic column made of silica bonded with a macrocyclic peptide, teicoplanin aglycone. With this stationary phase, two additives (trifluoroacetic acid and isopropylamine) were tested under extreme concentration conditions to observe the behavior of this peptide. Indeed, concentrations exceeding 1 M in the methanol co-solvent (>0.1 M overall concentration in the CO2-methanol mixture) were used whereas usual additive concentrations employed in supercritical fluid chromatography (SFC) rarely exceed 50 mM in the co-solvent. One purpose was to modify the apparent pH of the fluid, which is normally slightly acidic (around 5) and consequently possibly changing the ionization state of the peptide. Firstly, the effect of acidic and basic additives on the polarity and the apparent pH were evaluated with the help of color indicators. This served to assess the ionization state of the peptide under the selected operating conditions. Secondly, 54 achiral and 24 chiral molecules were injected in the chromatographic column at different levels of additives. The achiral species served at establishing retention models based on linear solvation energy relationships (LSER), while the chiral species were examined for their enantioresolution. From the LSER equations and observation of chromatograms, it appeared that specific interactions between the peptide-based stationary phase and the analytes evolved when increasing the concentration of additives, particularly hydrogen bonds and ionic interactions. A bare silica stationary phase (Acquity BEH) served as reference to deconvolute the contributions of silica support and bonded peptide. This study, with these extreme conditions of mobile phase, could be useful to understand the behavior of such peptides in SFC mobile phases and also improve the knowledge of the effects of additives in SFC, which should be helpful in the future prospect of analyzing large biomolecules in SFC.

Surface Design of Enantiomeric HPLC Separation on Vancomycin and Teicoplanin-Based Stationary Phases, a Tool for Chiral Recognition of Model ß-Blockers.

A quality-by-design approach was adopted for enantioseparation of atenolol on Vancomycin and Teicoplanin-based chiral stationary phases using reversed phase (RP) mode and polar ionic mode (PIM), respectively to account for major forces involved in enantiorecognition of ß-blockers on macrocyclics. A fractional factorial screening design for the two modes; followed by a central composite optimization design and regression analysis were able to point out critical factors and chromatographic responses and robust surface of the design. Within the studied range of flow the optimal was 0.3 mL/min for Chirobiotic T and 1 mL/min for Chirobiotic V. In PIM, a composition of 100% methanol was mandatory to compromise between best separation and least retention with equal amounts of the acid and base modifiers for enantiomers of atenolol, as model drug in addition to metoprolol and pindolol as structurally related compounds for possible extrapolation of results on members of the same class. However, in RP mode, only triethylamine acetate was needed as buffer for atenolol enantiomers. Chiral recognition of atenolol in both elution modes, further confirmed via extrapolation of the models on the two other ß-blockers showed that ionic interactions rather than any other forces governed chiral recognition on the two macrocyclic stationary phases in both modes.

Multicomponent antibiotic substances produced by fermentation: implications for regulatory authorities, critically ill patients and generics.

Teicoplanin and polymyxin E (colistin) are antibiotics consisting of multiple, closely related subcomponents, produced by fermentation. The principal components comprise a complex mixture of chemically related, active substances (teicoplanin A(2-1)-A(2-5) and polymyxin E(1-2), respectively), which might be required to be present in specific ratios to ensure optimal antibacterial and clinical efficacy. These subcomponents differ in their fatty acid and amino acid composition and, as such, the lipophilic and protein binding characteristics differ between components. This has therapeutic implications for critically ill patients, as the volume of distribution of the teicoplanin A2 and polymyxin E analogues at the onset of an intravenous infusion may impact on expected pharmacokinetics and influence outcome.

Synthesis of teicoplanin-modified hybrid magnetic mesoporous silica nanoparticles and their application in chiral separation of racemic compounds.

Teicoplanin-conjugated mesoporous silica magnetic nanoparticles (TE-MSMNPs) were fabricated as novel chiral magnetic nano-selectors. Successful preparation of the functional magnetic mesoporous materials was achieved by grafting teicoplanin on N-(2-aminoethyl)-3-aminopropyltrimethoxysilane-modified mesoporous silica Fe3O4 magnetic nanoparticles (AEAPTMS-MSMNPs), and this was confirmed by various characterization techniques. The synthesized magnetic nanoparticles were regularly spherical and uniformly mesoporous with an average diameter of around 600 nm and a mean pore size of about 3.9 nm, respectively. These versatile magnetic nanoparticles were effective in a direct chiral separation of five racemic compounds in phosphate buffer. Much stronger interactions were observed with the (+)-enantiomers than with the (-)-enantiomers. After washing with water and ethanol by sonication, TE-MSMNPs could be reused at least three times with little efficiency loss. The functional magnetic mesoporous nanoparticles were easily separated from the racemic solutions using an external magnetic field. These magnetic nano-materials are suitable for enantiomer separations.

Elimination of teicoplanin by adsorption to the filter membrane during haemodiafiltration: screening experiments for linezolid, teicoplanin and vancomycin followed by in vitro haemodiafiltration models for teicoplanin.

Pharmaceutical agents directed against methicillin-resistant Staphylococcus aureus can be eliminated during haemodiafiltration, not only by diffusion and ultrafiltration, but also by adsorption onto haemofilters. The latter may be affected by the binding of agents to serum albumin. The present study therefore investigated the affinity of anti-methicillin-resistant Staphylococcus aureus agents (teicoplanin, linezolid, vancomycin) for haemofilters and the pharmacokinetic properties of teicoplanin during haemodiafiltration. Linezolid, teicoplanin and vancomycin were first screened for their in vitro affinity for three different kinds of filter membranes: polysulfone, polyacrylonitrile and polymethylmethacrylate. Only teicoplanin showed significant filter-binding activity. An in vitro haemodiafiltration circulation model was then developed that incorporated a one-litre beaker containing Krebs-Ringer's bicarbonate solution with/without human albumin (0 or 3 g/dl) as an artificial plasma. Teicoplanin (initial concentration 50 µg/ml, representing the maximum plasma concentration (Cmax) resulting from a typical clinical dosage) was circulated throughout the beaker. Teicoplanin concentrations in the 'plasma' and ultrafiltrate were determined by high performance liquid chromatography. In the screening experiment, teicoplanin was predominantly adsorbed onto polysulfone and polymethylmethacrylate membranes. Furthermore, teicoplanin was primarily eliminated by adsorption onto these filters during in vitro haemodiafiltration. Albumin significantly reduced both haemodiafiltration clearance and the adsorption-dependent elimination, although there were complex but significant interactions between albumin and the filter membrane. Elimination of teicoplanin in an in vitro haemodiafiltration model was largely due to adsorption onto polysulfone and polymethylmethacrylate haemofilters. Future clinical studies should likely be designed to evaluate present recommendations of teicoplanin dosages in patients on haemodiafiltration.

Comparison of teicoplanin disk diffusion and broth microdilution methods against clinical isolates of Staphylococcus aureus and S. epidermidis

The CLSI M100-S19 document has recommended the disuse of vancomycin disks for staphylococci and informed that studies on the action of teicoplanin in disk-diffusion testing should be performed. We describe the comparison of two methods, disk diffusion and broth microdilution, for determining teicoplanin susceptibility in clinical isolates of staphylococci. Overall results showed an aggregation rate of 96.8 percent; Staphylococcus aureus showed total agreement while S. epidermidis showed 93.8 percent of agreement. According to these local results, disk diffusion can still be employed to teicoplanin susceptibility determination for staphylococci in our institution.

New chiral and restricted-access materials containing glycopeptides as selectors for the high-performance liquid chromatographic determination of chiral drugs in biological matrices.

Two new chiral and restricted-access materials containing glycopeptide antibiotics as chiral selectors (chiro-Glyco-RAM) were designed, suitable for the direct HPLC injection of biological fluids containing chiral drugs without any sample pre-treatment or pre-columns coupling. The external surface of the porous silica support was covered with a bio-compatible hydrophilic polymeric network (polyvinyl alcohol, PVA) while the chiral phase based on either teicoplanin (TE) or teicoplanin aglycone (TAG) was exclusively confined to the internal region. The chiro-Glyco-RAM supports were synthesized by the following steps: (a) introduction of 3-aminopropyl groups on 100 A pore size silica gel; (b) activation of the aminopropylated silica with 1,6-diisocyanatohexane; (c) functionalization of the external region of the porous silica with PVA; (d) covalent linking of TE/TAG to the internal surface. The average pore diameter of the chiro-Glyco-RAM supports, calculated by inverse size-exclusion chromatography (ISEC), was about 80 A and able to exclude macromolecules heavier than about 20,000 Da (such as the most abundant serum proteins) from the pores. The recovery of bovine serum albumin (BSA) was almost quantitative. HPLC analyses of model chiral drugs were performed using hydro-organic mobile phases consisting of an organic solvent (acetonitrile or methanol) and aqueous solutions of ammonium acetate (0.020 M) or ammonium formate (0.0025-0.0050 M).

Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC.

This paper deals with the chiral separation of triiodothyronine (T3) and thyroxine (T4) by HPLC and micro-HPLC. The separation of T3 and T4 is of great pharmaceutical and clinical interest, since the enantiomers exhibit different pharmacological activities. The HPLC measurements were performed on a chiral stationary ligand-exchange phase using l-4-hydroxyproline bonded via 3-glycidoxypropyltrimethoxysilane to silica gel as a selector. Also a chiral teicoplanin (Chirobiotic) phase was used. In micro-HPLC the chiral separation behaviour of l-4-hydroxyproline, and of the macrocyclic antibiotics teicoplanin and teicoplanin aglycone was investigated for the enantioseparation of T3 and T4. l-4-Hydroxyproline was bonded to 3 microm and the glycopeptide antibiotics were bonded to 3.5 microm silica gel and separations were accomplished by microbore HPLC columns (10 cmx1 mm I.D.). With both techniques and all chiral selectors investigated T3 and T4 were baseline resolved. micro-HPLC was found to be superior to analytical HPLC with respect to low consumption of packing material, mobile phase and analyte.

Could linear solvation energy relationships give insights into chiral recognition mechanisms? 1. Pi-pi and charge interaction in the reversed versus the normal phase mode.

Linear solvation energy relationships (LSER) have been used for years in liquid chromatography to access the factors that lead to retention and, more recently, to selectivity. In chiral separations, two enantiomers will receive exactly the same descriptors correctly predicting that they will not be separated by any non-chiral stationary phase. However, LSER studies could be used considering that each enantiomer sees a chiral stationary phase (CSP) differently. Working with the enantioselectivity factor, k'2lk'1 could give interesting information on the chiral recognition mechanisms. The b and v system parameters always predominantly contribute to a solute's retention in reversed phase liquid chromatography (RPLC) compared to the a, e and s parameters. However, these minor parameters for retention may become the essential ones for enantiomeric separations where a minimum of three simultaneous interactions is needed for an enantioseparation to occur. In this non-chiral study, six different stationary phases, a classical C(18), a diphenyl bonded stationary phase (DP), a polystyrene-divinylbenzene (DVB), a polar embedded new stationary phase, an anion exchanger (SAX) and a teicoplanin aglycone (TAG) CSP were studied with achiral test solutes. Significantly higher e terms were obtained for the SAX and TAG columns. It seems that the ability of stationary surface charges to induce dipoles in polarizable molecules is encoded mainly in the e term. Since the DP and DVB columns did not produce e parameters significantly higher than the C(18) column, it seems that pi-pi interactions are (a) extremely weak in RPLC or (b) they are not simply encoded in this single e system parameter but spread in at least three parameters. The TAG CSP produced logically very different parameters when used in the reversed phase mode compared to the normal phase mode showing the critical role of the mobile phase.

Comparisons of the separations of some neutral analytes by LC, MEKC, and CEC.

The relative utility of high-performance liquid chromatography, micellar electrokinetic chromatography (MEKC), and capillary electrochromatography (CEC) is examined for the separation of essentially uncharged solute mixtures. Three model systems are used for which separations by reversed-phase liquid chromatography had been established. These consisted of a set of three substituted hydroxybenzoates; a mixture of six structurally closely related steroids; and the multicomponent aminoglycoside antibiotic, teicoplanin. These sets represented a range of difficulty in achieving separations by reversed-phase LC. It was found that equivalent or better separations for all systems could be established by MEKC and CEC. Both electrophoretic techniques offer much higher peak efficiencies than LC, and MEKC is found to be superior to CEC in terms of peak efficiencies and ruggedness of operation.

Isolation and structure determination of a novel complex of the teicoplanin family.

A new teicoplanin-like antibiotic was discovered when using Actinoplanes teichomyceticus strain 3/W, the fermentation medium containing Alburex, and the fermentation time 275 hours. The new product was separated from teicoplanin complex by polyamide resin chromatography and purified by Amberlite XAD-7 and affinity resin chromatographies. The structure was established on the basis of the physico-chemical characteristics of the complex and of its aglycone. The new structure is that of teicoplanin with a carbonyl group substituting for the CHNH2 group of amino acid 1. We hypothesize that the novel complex is a transformation product of teicoplanin due to a simple transamination reaction, as supported by its structure and by the concomitant decrease in teicoplanin concentration during its production.