Ultraselective antibiotic sensing with complementary strand DNA assisted aptamer/MoS2 field-effect transistors.

Although aptamer has been demonstrated as an important probe for antibiotic determination, the selective sensing of different antibiotics is still a challenge due to their structure similarities and wide folding degrees of aptamer. Herein, a field-effect transistor using MoS2 nanosheet as the channel and an aptamer DNA (APT) with its configuration shaped by a complementary strand DNA (CS) is employed for kanamycin (KAN) determination. This probe structure contributes to an enhanced selectivity and reliability with reduced device-to-device variations. This MoS2/APT/CS sensor shows time-dependent performance in antibiotic sensing. Prolonged detection time (20 s-300 s) leads to an enhanced sensitivity (1.85-4.43 M-1) and a lower limit of detection (1.06-0.66 nM), while a shorter detection time leads to a broader linear working range. A new sensing mechanism relying on charge release from probe is proposed, which is based on the "replacement reaction" between KAN and APT-CS. This sensor exhibits an extremely high selectivity (selectivity coefficient of 12.8) to kanamycin over other antibiotics including streptomycin, tobramycin, amoxicillin, ciprofloxacin and chloramphenicol. This work demonstrates the merits of probe engineering in label-free antibiotic detection with FET sensor, which presents significant promises in sensitive and selective chemical and biological sensing.

Ultratrace antibiotic sensing using aptamer/graphene-based field-effect transistors.

Antibiotic residue, as emerging pollution resulting from antibiotic abuse, poses a serious threat on ecosystem and human health. Conventional methods for antibiotic detection, e.g., liquid/gas chromatography, are based on complicated instruments and time-consuming; therefore, efforts have been made to realize in situ and real-time monitoring of antibiotics. Here, a miniaturized and integratable electronic antibiotic sensor based on field-effect transistor (FET) is reported. The reduced graphene oxide (rGO) nanosheet is used as the channel material and the aptamer RNA for tobramycin is modified onto rGO as the probe. A novel sensor design with 6-mercapto-1-hexanol (MCH)/1-pyrenebutanol (PBA) blocking layer (BL) for structure optimization is applied to enhance the sensor reliability and specificity. This rGO/aptamer/BL sensor shows an ultra-sensitivity to tobramycin with a lower detection limit of 0.3 nM and a quick response within 5 s, as well as a high specificity over other antibiotics such as kanamycin, streptomycin, ciprofloxacin, and tetracycline. The sensing mechanism based on the deformation of the charged aptamer probe is proposed via an in-depth analysis of the interactions between aptamer, tobramycin and rGO. In addition, sensing test performed under controlled microfluidic flow conditions demonstrates a great potential of the sensors in practical applications.

Electrochemical biosensing based on protein-directed carbon nanospheres embedded with SnOx and TiO2 nanocrystals for sensitive detection of tobramycin.

A series of nanocomposites comprised of homogeneous mesoporous carbon nanospheres embedded with SnOx (x = 0, 1, or 2) and TiO2 nanocrystals using bovine serum albumin (BSA) as template followed by calcinated at different temperatures (300, 500, 700, and 900°C) were prepared, and were denoted as SnOx@TiO2@mC. Then a novel electrochemical biosensing strategy for detecting tobramycin (TOB) based on the nanocomposites was constructed. The as-prepared SnOx@TiO2@mC nanocomposites not only possess high specific surface area and good electrochemical activity but also exhibit strong bioaffinity with the aptamer strands, therefore, they were applied as the scaffold for anchoring TOB-targeted aptamer and further used to sensitively detect trace TOB in aqueous solutions. By comparing the electrochemical biosensing responses toward TOB detection based on the four SnOx@TiO2@mC nanocomposites, the biosensing system constructed with SnOx@TiO2@mC900 (derived at 900°C) demonstrated the highest determination efficiency, high selectivity, and good stability. In particular, the new proposed aptasensing method based on SnOx@TiO2@mC nanocomposite exhibits considerable potential for the quantitative detection of TOB in the biomedical field.

Urea-formaldehyde monolithic column for hydrophilic in-tube solid-phase microextraction of aminoglycosides.

A novel urea-formaldehyde (UF) monolithic column has been developed and exploited as a sorbent for hydrophilic in-tube solid-phase microextraction (in-tube SPME) of aminoglycosides (AGs). Because of the innate hydrophilicity, UF monolith showed high extraction efficiency towards these hydrophilic analytes. The adsorption capacities for target compounds dissolved in water/ACN (1:1, v/v) were in the range of 5.18-7.36µg/cm. Due to the lack of a chromophore, evaporative light scattering detector (ELSD) was selected as the detector for AGs, and coupled with the online in-tube SPME-HPLC system. Several factors of the online system, such as trifluoroacetic acid (TFA) and ACN percentage in the sampling solution, ionic strength in the sample solution, elution volume, sampling and elution flow rate, were optimized with respect to the extraction efficiencies. Under the optimized conditions, the limits of detection (LODs) of streptomycin, tobramycin and neomycin were discovered in the range of 3.0-5.2µg/kg. The recoveries were ranged from 82.1 to 96.7% with relative standard deviations (RSDs) of 2.3-5.1% (n=4) at spiking levels of 50, 200 and 500µg/kg, respectively. The excellent applicability of the UF monolithic column was examined by the determination of streptomycin in practical tilapia samples, which showed the potential advantages for the analysis of polar analytes in complicated samples.

Micellar Electrokinetic Chromatography of Aminoglycosides.

The components of the aminoglycosides, e.g., gentamicin, sisomicin, netilmicin, kanamycin, amikacin, and tobramycin, and related impurities of these antibiotics can be separated by means of micellar electrokinetic chromatography (MEKC). Derivatization with o-phthaldialdehyde and thioglycolic acid is found to be appropriate for these antibiotics. The background electrolyte was composed of sodium tetraborate (100 mM), sodium deoxycholate (20 mM), and ß-cyclodextrin (15 mM) having a pH value of 10.0. This method is valid for evaluation of gentamicin, kanamycin, and tobramycin. It has to be adopted for amikacin, paromomycin, neomycin, and netilmicin.

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate.

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.

HPLC retention behavior on hydride-based stationary phases.

Two stationary phases attached to a silica hydride surface, cholesterol and bidentate C18, are investigated with a number of pharmaceutically related compounds in order to illustrate the various retention mechanisms that are possible for these bonded materials. The test solutes range from hydrophilic to hydrophobic based on log P (octanol/water partition coefficient) and pKa values. The mobile phases consist of acidified (formic and perchloric acid) water/methanol or water/ACN mixtures. Of particular interest are the high organic content mobile phase compositions where the retention would increase if the bonded material was operating in the aqueous normal phase (ANP) mode. Plots of retention factor (k) versus mobile phase composition are used to elucidate the retention mechanism. A number of examples are presented where solutes are retained based on RP, ANP, or dual retention mechanisms. The silica hydride-based stationary phases can also retain compounds in the organic normal phase.

Rapid and selective micellar electrokinetic chromatography for simultaneous determination of amikacin, kanamycin A, and tobramycin with UV detection and application in drug formulations.

A simple and selective micellar electrokinetic chromatography (MEKC) with UV detection is described for simultaneous determination of amikacin, tobramycin, and kanamycin A, performed in Tris buffer (180 mM; pH 9.1) with 300 mM sodium pentanesulfonate (SPS) as an anionic surfactant. Under this condition, good separation with high efficiency and the required short analysis time is achieved. The linear ranges of the method for the determination of amikacin, tobramycin, and kanamycin A were 0.1-0.5 mg / mL, 0.4-2.0 mg / mL, and 0.4-2.0 mg / mL, respectively; the detection limits (signal-to-noise ratio = 3; injection, 0.5 psi 5 s) were 0.08, 0.2, and 0.2 mg / mL, respectively. The small amount of sample required and the expeditiousness of the procedure allow content uniformity to be determined in individual commercial products.

[A novel laboratory method of isolation and purification of tobramycin]. / Razrabotka novogo laboratornogo metoda vydeleniia i ochistki tobramitsina.

A new laboratory method for isolation and purification of tobramycin by using extraction of a tobramycin derivative with benzaldehyde by methylene chloride, subsequent hydrolysis of azomethine and recrystallization of the formed tobramycin sulfate from solution of sulfuric acid in methanol was developed. The method allows to exclude the stage of chromatographic purification of tobramycin, to reduce the time of the process realization from 120-125 h to 15-20 h, to increase the yield of the target product from 37-40% to 60-65% without decreasing the product quality, to exclude a number of large-size and expensive equipment and to ensure high reproducibility of the technology.

Development and validation of capillary electrophoresis method for tobramycin with precapillary derivatization and UV detection.

One of the major drawbacks in the analysis of aminoglycoside antibiotics is their lack of UV chromophore and/or fluorophore. Tobramycin, a representative member of this group, was examined in this study. To overcome the detection hurdle, a precapillary derivatization followed by capillary electrophoresis analysis with direct UV detection was investigated. A central composite design was applied to optimize the method and three parameters were selected in this study: buffer pH, temperature and % acetonitrile (ACN). Selectivity between tobramycin main component and its adjacent peaks as well as the peak efficiency and symmetry factors were established as responses. For each response, a model was obtained by a second-order mathematical expression. Successful results were obtained with a simple background electrolyte (BGE) containing 30 mM sodium tetraborate, pH 10.2, and ACN (75:25 v/v). Under these conditions, baseline separation of tobramycin from its adjacent kanamycin B and an unknown peak was achieved. A temperature of 20 degrees C and applied voltage of 28.0 kV were used. The method showed good validation data in terms of precision, limits of quantitation and detection, specificity and linearity and was found to be suitable for analysis of tobramycin bulk pharmaceutical samples.

[Quantitative determination of the antibiotic tobramycin using high-performance liquid chromatography]. / Kolichestvennoe opredelenie antibiotika tobramitsina s pomoshch'iu vyskooéffektivnoi zhidkostnoi khromatografii.

Separation of the main components of the nebramycin complex of apramycin, kanamycin B and tobramycin was achieved in the form of their 2,4-dinitrophenyl derivatives. A chromatograph SR 8000 of 'Spectra-Physics" and a column 'Sperisorb C6" (4.6 X 250 mm) with the granule size of 5 micrometers were used in the study with the mobile phase of acetone-trisbuffer, pH 7 in a ratio of 65 to 35, the flow rate of 1 ml/min, a temperature of 35 degrees C and detection at lambda 350 nm. Quantitative determination of the tobramycin purity level was performed with an equation developed for the given conditions. The purity levels of the reference tobramycin base and its sulfate were estimated. The results of the estimation corresponded to the data of biological titration.

Liquid-chromatographic determination of tobramycin in serum with spectrophotometric detection.

We describe a simple, precise, accurate, and specific liquid-chromatographic procedure for determination of tobramycin in 50 microL of serum. Tobramycin and the internal standard (sisomicin) are quantitatively converted into their trinitrophenyl derivatives by reaction with a water-soluble derivatizing agent (2,4,6-trinitrobenzenesulfonic acid) at 70 degrees C for 30 min. The derivatives are extracted from the crude reaction mixture by using a reversed-phase Bond-Elut C18 column, and separated on a reversed-phase octyl column with a mobile phase consisting of an acetonitrile/phosphate buffer (70/30 by vol) at a flow rate of 3.0 mL/min. The eluted compounds are detected at 340 nm, and quantified from their peak areas. Chromatography is complete in less than 4.5 min at the optimum column temperature of 50 degrees C. The lower limit of detection for tobramycin is less than 0.2 mg/L. Analytical recoveries for tobramycin varied from 94 to 99%, linearity extended to 25 mg/L, and day-to-day precision (CV) was between 4.6 and 5.1%. Numerous drugs and antibiotics tested do not interfere. Results correlate well (r greater than 0.95) with those by radioimmunoassay and EMIT.

[Comparative characteristics of the methods for assessing aminoglycoside extraction with the artificial kidney tobramycin clearance and dialyzability]. / Sravnitel'naia kharakteristika metodov otsenki ékstraktsii aminoglikozidov apparatom "iskusstvennaia pochka": klirens i dializuemost' tobramitsina.

The pharmacokinetics of tobramycin after its intravenous or intramuscular injection in a dose of 80 mg for 60 minutes was studied in 8 patients with chronic glomerulonephritis in the terminal stage of chronic renal insufficiency. The drug levels wee determined in the arterial (CA) and venous (CV) blood and dialyzates (CD) during the hemodialysis (6 hours) and 13-70 hours before the hemodialysis. The antibiotic was administered simultaneously with connection of the "artificial kidney" apparatus (KIIL) or 1 hour after it. The values of the clearance (CID) and dialyzing (D) of tobramycin were calculated with the following equations: (CID)1 equals Q(CA minus CV)/CA; (CID)4 equals FCD/CA; (D)2 equals Q(CA minus CV)/(CA minus CD); (D)5 equals FCD/(CA minus CD), where Q and F are the rates of the blood and dialysate flow respectively. In all cases the values of CID and D correlated and the difference between them was not significant. During the hemodialysis the values of (CID)1 varied to a greater extent than those of (CID)4. Irrespective of the procedure for estimation of CID the above variation was not pronounced, when tobramycin was administered simultaneously with initiation of the hemodialysis or during it than long before connection of the "artificial kidney" apparatus. In this connection it is recommended that antibiotic extraction be characterized by determination of (CID)4 on the drug administration long before the initiation of the hemodialysis. When Q equals 200 ml/min and F equals 600 ml/min, the average value of CLD for tobramycin was equal to 64 ml/min and the extraction coefficient was equal to 35 per cent.