Irena Choma

Bioautography detection in thin-layer chromatography

Bioautography is a microbial detection method hyphenated with planar chromatography techniques. It is based mainly on antimicrobial or antifungal properties of analyzed substances. The review discusses three versions of bioautography, i.e. contact, immersion and direct bioautography. The more concern is given to the last one. Many applications are quoted, not only for testing various groups of compounds, but also for investigating biochemical processes and factors influencing bacterial growth. Additionally, related methods, which can be included into direct bioautography, are discussed. The most promising among them seems to be TLC-bioluminescence screening.

TLC separation of fluoroquinolones: Searching for better selectivity

Abstract Fluoroquinolones are a relatively new group of chemotherapeutics used worldwide. They are approved for use as therapeutic agents and as feed additives in food producing animals in many countries. The misuse of these antibiotics in human and veterinary medicine has led to the loss of their efficacy and to the emergence of drug‐resistant bacteria. Therefore, there is a need for developing analytical methods to monitor the levels of fluoroquinolone residues in biological fluids and edible animal tissues. In this paper, the optimal conditions for thin‐layer chromatography (TLC) analysis of six veterinary fluoroquinolones, i.e., difloxacin, ciprofloxacin, norfloxacin, sarafloxacin, enrofloxacin, and flumequine are established. Good separation was achieved using 2‐dimensional TLC on silica gel. Retention parameters for various chromatographic systems are compared. Chromatographic properties of four sorbents, i.e., plane silica gel and silica gel with bonded diol‐, amino‐, and cyanopropyl chains are discussed.Abstract Fluoroquinolones are a relatively new group of chemotherapeutics used worldwide. They are approved for use as therapeutic agents and as feed additives in food producing animals in many countries. The misuse of these antibiotics in human and veterinary medicine has led to the loss of their efficacy and to the emergence of drug‐resistant bacteria. Therefore, there is a need for developing analytical methods to monitor the levels of fluoroquinolone residues in biological fluids and edible animal tissues. In this paper, the optimal conditions for thin‐layer chromatography (TLC) analysis of six veterinary fluoroquinolones, i.e., difloxacin, ciprofloxacin, norfloxacin, sarafloxacin, enrofloxacin, and flumequine are established. Good separation was achieved using 2‐dimensional TLC on silica gel. Retention parameters for various chromatographic systems are compared. Chromatographic properties of four sorbents, i.e., plane silica gel and silica gel with bonded diol‐, amino‐, and cyanopropyl chains are dis...

TLC-DB as an Alternative to the HPLC Method in the Determination of Cefacetril Residues in Cow's Milk

Abstract Cephalosporins, relatively new antibiotics related to penicillins, are widely used in the treatment of both human and veterinary diseases because of their broad spectrum of antibacterial activity and good pharmacokinetic properties. Cefacetril, belonging to cephalosporins, is commonly used in treating mastitis in cows. In the present paper, cefacetril excretion with milk was examined by means of the TLC-DB and HPLC method. Thin-layer chromatography-direct bioautography is the technique which combines TLC with microbiological detection. Semi-quantitative determination of cefacetril in milk by TLC-DB was compared with quantitative HPLC analysis. An exponential relationship was proposed for calibration curves in bioautography.

Semiquantitative Estimation of Enrofloxacin and Ciprofloxacin by Thin‐Layer Chromatography–Direct Bioautography

Abstract Fluoroquinolones, relatively new chemotherapeutics, are widely used in the treatment of both human and veterinary diseases because of their broad spectrum of antibacterial activity and good pharmacokinetic properties. Enrofloxacin is used in veterinary medicine, while its main metabolite, ciprofloxacin, is one of the most frequently used human antibiotics in the world. Hence, there is a need for assays capable of determining and distinguishing between these drugs. Thin‐layer chromatography–bioautography (TLC–B) combines TLC with microbiological detection. In the present paper, semiquantitative determination of enrofloxacin and ciprofloxacin standards by TLC–B using Chrom Biodip® Antibiotics Test Kit is presented. The optimal conditions for bioautographic detection were established. The exponential relations were proposed for approximation of dependencies between areas of zone inhibitions and logarithm of the amounts applied. The detection limit for standards of both enrofloxacin and ciprofloxacin was 0.01 ppm, lower than maximum residue limits (MRLs) established for various food products.

Thin-Layer Chromatography - Direct Bioautography for the Screening of Antimicrobial Properties of Plant Extracts

The main volatile compounds from three medicinal plants belonging to Lamiaceae family were screened for their biological properties. The plants were Salvia officinalis, Thymus vulgaris, and Mentha × piperita containing as the main volatile constituents thujone, thymol, and menthol, respectively. The applied chromatographic system was silica gel developed with toluene-ethyl acetate (93:7). Thin-layer chromatography — direct bioautography (TLC-DB) against Escherichia coli and Bacillus subtilis was used for detection of antibacterial activity of the plant extracts and essential oils. The bioautographic fingerprints were compared with the fingerprints obtained after derivatization with anisaldehyde.

Matrix Solid‐Phase Dispersion Combined with Thin‐Layer Chromatography–Direct Bioautography for Determination of Enrofloxacin and Ciprofloxacin Residues in Milk

Abstract Enrofloxacin is a common veterinary antibiotic, which can be found as a residue in milk, together with its main metabolite, ciprofloxacin. Chromosorb WAW was used as a sorbent for matrix solid‐phase dispersion of milk samples spiked with these antibiotics. This pre‐separation method was combined with thin‐layer chromatography‐direct bioautography to obtain semi‐quantitative results. Various modes of the procedure were tested and the one giving the best recovery of the antibiotics from milk was chosen.

Screening of Enrofloxacin and Ciprofloxacin Residues in Milk by HPLC and by TLC with Direct Bioautography

Fluoroquinolones are a relatively new group of synthetic antibiotics (chemotherapeutics) widely used both in hu man and animal treatment. Because veterinary enrofloxacin and its main metabolite ciprofloxacin, a widely used hu man antibiotic, can be present as residues in milk, there is a need for methods for their separation and determination. Matrix solid-phase dispersion (MSPD) on a siliceous sorbent was used for isolation and concentration of enrofloxacin and ciprofloxacin residues from cows’ milk. This preseparation method was combined with high-performance liquid chromatography and with thin-layer chromatography-direct bioautography. Mean recoveries were calculated for different levels of the antibiotics in milk. The results revealed that thin-layer chromatography-direct bioautography can be used for the screening of enrofloxacin and ciprofloxacin at the maximum residue level stipulated for milk by the European Union.

High-performance liquid chromatography of benzodiazepines using sorbents with thermally immobilized Carbowax 20M

The use of siliceous supports (controlled-porosity glass or silica gel) with thermally immobilized Carbowax 20M as sorbents for the separation of benzodiazepines by high-performance liquid chromatography (HPLC) was studied. The physico-chemical and chromatographic properties of these sorbents were compared with those of LiChrosorb DIOL and LiChrosorb RP-18. The results indicate that normal-phase HPLC chromatography of benzodiazepines using sorbents with thermally immobilized Carbowax 20M is a simple and effective method characterized by a simple binary mobile phase, very short column equilibration, high selectivity and a short time of analysis.

Separation, Identification, and Investigation of Antioxidant Ability of Plant Extract Components Using TLC, LC–MS, and TLC–DPPH•

Ten common phenolic compounds from the extracts of five plant species, that is, Hypericum perforatum L., Matricaria recutita L., Achillea millefolium L., Thymus vulgaris L., and Salvia officinalis L., were separated using optimized thin-layer chromatography (TLC) conditions at normal phase mode and were visualized using Natural Product/Polyethylene Glycol Reagent. Antioxidant properties of components of the extracts were assessed using TLC–DPPH• directly on the TLC plate. Nine out of ten analyzed polyphenols were proved to be radical scavengers. The presence and identity of target substances in plant samples were confirmed by the liquid chromatography–mass spectrometry technique.

A Novel Application of Matrix Solid‐Phase Dispersion for Determination of Doxycycline and Flumequine Residues in Milk

Abstract A novel version of matrix solid‐phase dispersion (MSPD) for isolation of doxycycline (DC) and flumequine (FL) in milk was established. The MSPD procedure involves blending of sample with solid support material, usually silica with bonded C18 chains. In the work presented, bare siliceous sorbents were applied i.e., kieselguhr, wide‐pore silica gel and Chromosorb WAW. The sorbents were blended with the spiked milk using sorbent to milk ratio 4:1 w/w. The “pastes,” formed in that way, were put into the syringe and defatted with hexane. Then, dichloromethane was used to elute FL. The mixture of 0.5 M citric acid/methanol/2‐propanol (1:3:1) or 0.5 M citric acid/methanol (1:9) were used to elute DC. RP‐HPLC and NP‐HPLC were used for simultaneous determination of the antibiotics. It was possible to detect the antibiotics in milk at 100 ppb level. Chromosorb proved to be the optimal MSPD sorbent.