Concentration-dependent photoluminescence carbon dots for visual recognition and detection of three tetracyclines.

Concentration-dependent photoluminescence carbon dots (CDs) have been successfully synthesized through the one-step hydrothermal treatment of o-phthalic acid and ethylenediamine. The CDs possessed higher fluorescence quantum yield, up to 39.22%, exhibiting distinguished optical property, water solubility, and stability. The CDs that emit strong blue-green fluorescence can visually identify and determine tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC). TC quenched the fluorescence of CDs at 500 nm owing to the inner filter effect; OTC behaved similarly, but the emission wavelength of CDs was red-shifted to 515 nm. Inversely, once CTC was introduced to CDs solution, the fluorescence increased and the emission peak was blue-shifted to 450 nm. Bandgap transition and electrostatic interaction were proposed to be the mechanisms for the detection of OTC and CTC by CDs. Wide linear relationships were established for TC, OTC, and CTC with the limits of detection to be 50 nM, 36 nM, and 373 nM, respectively. Furthermore, the nanoscale probe constructed by this system has been applied to detect tetracyclines (TCs) in complex samples with satisfying recoveries (93.2-114%) and was designed as a portable test strip sensor for visually on-site TCs of honey sample screening. Accordingly, the preparation process of the nano fluorescent probe is simple and environmentally friendly, and the probe has a specific recognition ability for tetracyclines. The synthesized CDs in this work provide a new orientation for fast, effective, and visual real-time detection of tetracycline in actual samples.

The search for a microbiological inhibition method for the rapid, broad-spectrum and high-throughput screening of six kinds of antibiotic residues in swine urine.

In this study, a microbiological inhibition method for rapidly screening antibiotics in swine urine was established with an easy sample pre-treatment. The microbiological system consisted of an agar medium mixed with nutrients, sensitizers, a test bacterium (Geobacillus stearothermophilus ATCC12980) and pH indicator (bromocresol purple). It was observed that the detection limits of the test kit for twenty-eight common antimicrobial residues in urine, including ß-lactams, aminoglycosides, tetracyclines, sulfonamides, macrolides, and lincosamides, were less than or equal to the maximum residue limits of the kidney, as determined by the EU and China. Moreover, the false negative rate and the false positive rate, along with other performance indexes such as interassay coefficients of variation and shelf life of the kit, all met the standard requirements of the ISO13969:2003 guidelines. Additionally, our results were consistent with those using the gold-standard physical chemistry method, which suggest the proposed method is suitable for screening antibiotic residues.

Challenges in the bioanalysis of tetracyclines: Epimerisation and chelation with metals.

Tetracyclines (TCs) are important broad spectrum antibiotics which are active against gram-positive and gram-negative bacteria. TCs readily form epimers, especially under weakly acidic conditions. The epimers are reported to have different antibacterial and toxicological properties and pose a significant challenge for selective bioanalysis, being isobaric with the parent drug and possessing very similar physicochemical properties. During the development, validation and use of bioanalytical methods for minocycline in plasma, urine and renal dialysate there were two unexpected findings. The first was that the analyte and the internal standard, tetracycline, were found to be unexpectedly stable and resistant towards epimerisation in the presence of the deproteinising agent, trichloroacetic acid (TCA). The second was that keeping minocycline spiked dialysate in a freezer led to significant losses which were worse for low concentrations at lower storage temperatures. Investigations into the stability of tetracycline, minocycline, omadacycline and tigecycline in aqueous acidic solutions, under typical analytical conditions, revealed that TCA acts as a stabiliser with respect to both epimerisation and other degradation pathways for these TCs. This gives the rarely used TCA a significant advantage over the commonly used deproteinising agents such as acetonitrile when analysing TCs. Studies of the recoveries of tetracycline and tigecycline from frozen renal dialysis buffer demonstrated similar losses to those for minocycline. These were assigned to deposition of insoluble Mg2+ or Ca2+ complexes on freezing, as pre-storage treatment of the samples by incubation in EDTA coated tubes at room temperature prevented the losses. Minocycline was stable in renal dialysis buffer samples when frozen, for up to ca. 3 months, when this treatment was employed. The TCs were analysed using LC-MS/MS based methods developed in-house using the assay that was originally developed for minocycline in plasma, urine and dialysate as a template.

A highly fluorescent lanthanide metal-organic framework as dual-mode visual sensor for berberine hydrochloride and tetracycline.

A microscale highly fluorescent Eu metal-organic framework (Eu-MOF) was synthesized with terephthalic acid and 1H-1,2,4-triazole-3,5-diamine by one-pot hydrothermal method. And it was characterized by scanning electron microscope, Fourier transform infrared spectroscopy, powder X-ray diffraction, fluorescence spectroscopy, thermogravimetric analysis, and energy dispersive X-ray mapping. The prepared Eu-MOF has high quantum yield of 30.99%, excellent water dispersibility, good fluorescence stability, and favorable thermal stability. Based on the distinctly different fluorescence responses of different emission, the prepared Eu-MOF was used as dual-mode visual sensor for the sensitive detection of berberine hydrochloride and tetracycline. The limits of detection are 78 nM and 17 nM, respectively. The sensing mechanism was also discussed. Moreover, a filter paper sensor has been designed for sensing tetracycline with a notable fluorescence color change from blue to red. The prepared Eu-MOF is promising to be developed as a multi-mode luminescent sensor for visual detection in biochemical analysis. Graphical abstract Illustration of the synthesis of Eu-MOF and its sensing applications for berberine hydrochloride and tetracycline.

Pharmacokinetics, Safety, and Clinical Outcomes of Omadacycline in Women with Cystitis: Results from a Phase 1b Study.

Omadacycline, an aminomethylcycline antibiotic, is approved as once-daily intravenous (i.v.) and oral (p.o.) monotherapy for acute bacterial skin and skin structure infections and for community-acquired bacterial pneumonia, and it is under development for treatment of urinary tract infection (UTI). This is a phase 1b, randomized, open-label study of omadacycline in women with cystitis (defined as UTI symptoms and a positive urine leukocyte esterase test). Patients received omadacycline for 5 days (group 1: 200 mg intravenously on day 1, then 300 mg orally every 24 h [q24h]; group 2: 300 mg orally every 12 h [q12h] on day 1, then 300 mg orally q24h; group 3: 450 mg orally q12h on day 1, then 450 mg orally q24h). Blood and urine samples were collected over 5 days. Investigator-assessed clinical response was determined at end of treatment (EOT; day 6) and posttreatment evaluation (PTE; 5 to 9 days after last dosing). A total of 31 women were treated. At steady state (day 5), the range of mean omadacycline urine concentrations over 24 h across the groups was 17.94 to 48.12 µg/ml. The most common treatment-emergent adverse events were gastrointestinal (including nausea [60% to 73%] and vomiting [20% to 40%]) and were generally mild and transient. Investigator-determined clinical success was observed in 94% and 84% of patients at EOT and PTE, respectively, with similar results across groups. A favorable microbiological response at PTE was observed in 78% of patients who had a baseline pathogen. Omadacycline is partially excreted in urine and appears to be safe and well tolerated. These preliminary results indicate that omadacycline warrants further evaluation in larger controlled UTI studies.

Determination of tetracyclines in human urine samples by capillary electrophoresis in combination with field amplified sample injection.

A sensitive method using CZE-UV detection has been developed for the determination of five tetracycline antibiotics in human urine samples. To improve the sensitivity of the method, an on-line preconcentration strategy, named field-amplified sample injection, has been developed, based on the electrokinetic injection of the sample, which requires only a 1:100 dilution with sample solvent before injection. Under optimum conditions, sensitivity enhancement factors ranged from 450 to 800 for the studied compounds. The applicability of the proposed method was demonstrated by the determination of these antibiotics in spiked urine samples. The limits of quantification were lower than 0.8 mg/L and the precision (intra- and inter-day), expressed as %RSD was below 14%. Recoveries ranged from 92.1 to 96.7%. Thus, the proposed procedure is a simple, fast and efficient strategy which could be used as therapeutic drug monitoring in human urine samples.

Short-term toxicity assessments of an antibiotic metabolite in Wistar rats and its metabonomics analysis by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

4-Epi-oxytetracycline (4-EOTC), one of main oxytetracycline (OTC) metabolites, can be commonly detected in food and environment. The toxicity and effects of OTC on animals have been well characterized; however, its metabolites have never been studied systemically. This study aims to investigate 15-day oral dose toxicity and urine metabonomics changes of 4-EOTC after repeated administration in Wistar rats at daily doses of 0.5, 5.0 and 50.0mg/kg bw (bodyweight). Hematology and clinical chemistry parameters, including white blood cell count, red blood cell count, total protein, globulin and albumin/globulin, were obviously altered in rats of 5.0 and 50.0mg/kg bw. Histopathology changes of kidney and liver tissues were also observed in high-dose groups. Urinary metabolites from all groups were analyzed using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Seventeen metabolites contributing to the clusters were identified as potential biomarkers from multivariate analysis, including aminoadipic acid, 6-phosphogluconate, sebacic acid, pipecolic acid, etc. The significant changes of these biomarkers demonstrated metabonomic variations in treated rats, especially lysine and purine metabolism. For the first time in this paper, we combined the results of toxicity and metabonomics induced by 4-EOTC for the serious reconsideration of the safety and potential risks of antibiotics and its degradation metabolites.

Determination of veterinary antibiotics in bovine urine by liquid chromatography-tandem mass spectrometry.

A follow-up of antibiotics (tetracyclines, fluoroquinolones, cephalosporins, penicillins and amphenicols) in the bovine urine is important for two reasons: to understand if they are still present in organism, and whether their occurrence in urine might be considered as an environmental risk. A validated HPLC-MS/MS method (Decision 2002/657/EC) for antibiotics determination in bovine urine was developed. CCα and CCß were in the range of 0.58-0.83 and 0.55-1.1 ng mL(-1), respectively. Recoveries were 92-108%, with inter-day repeatability below 12%. Analysis of bovine urine revealed frequent presence of tetracyclines, which was related with animal's age. The cause, most presumably, might be found in different therapeutic protocols applied for veal calves and young bulls enrolled in this study. Most abundant was oxytetracycline with highest level in veal calves (1718 ng mL(-1)) vs. young bulls (2.8 ng mL(-1)). Our results indicate the necessity of antibiotics monitoring in bovine urine before animals undergo further processing in the food industry.

A new spectrofluorimetric method for the determination of some tetracyclines based on their interfering effect on resonance fluorescence energy transfer.

A rapid, simple, inexpensive and highly sensitive spectrofluorimetric method was developed for the determination of trace amounts of some tetracyclines (TCs), namely tetracycline hydrochloride (TCH), oxytetracycline hydrochloride (OTCH) and minocycline hydrochloride (MCH). Binding rhodamine B (RhB) to gold nanoparticles (Au NPs) resulted in quenching of the fluorescence of RhB by a resonance energy transfer (FRET) mechanism, with Au NPs as the energy acceptors. The presence of TCs caused the release of RhB molecules and recovered their fluorescence, and this was used as a basis for the quantitative determination of TCs. The reaction was monitored spectrofluorimetrically by measuring the increase in fluorescence of RhB at 572 nm starting 5 min after mixing the reagents in Tris buffer solution (pH 6.5). The effect of various experimental factors such as buffer type, pH, concentrations of the involved reagents and reaction time were studied to optimize the reaction conditions. Under optimum conditions, the calibration graphs were linear within the ranges 2.08 × 10(-9) -1.04 × 10(-6) mol/L, 2.01 × 10(-9) -1.00 × 10(-6) mol/L and 2.02 × 10(-9) -1.01 × 10(-6) mol/L and detection limits (LODs) of 0.61 × 10(-9) , 0.32 × 10(-9) and 0.66 × 10(-9) mol/L were calculated for TCH, OTCH and MCH, respectively, with corresponding percent relative standard deviations (%RSDs) of 1.18, 1.21 and 1.54 (n = 5). The method was successfully applied to the determination of TCs in drinking water, human urine, bovine milk and breast milk samples.

Antibiotic exposure in a low-income country: screening urine samples for presence of antibiotics and antibiotic resistance in coagulase negative staphylococcal contaminants.

Development of antimicrobial resistance has been assigned to excess and misuse of antimicrobial agents. Staphylococci are part of the normal flora but are also potential pathogens that have become essentially resistant to many known antibiotics. Resistances in coagulase negative staphylococci (CoNS) are suggested to evolve due to positive selective pressure following antibiotic treatment. This study investigated the presence of the nine most commonly used antimicrobial agents in human urine from outpatients in two hospitals in Ghana in relation to CoNS resistance. Urine and CoNS were sampled (n = 246 and n = 96 respectively) from patients in two hospitals in Ghana. CoNS were identified using Gram staining, coagulase test, and MALDI-TOF/MS, and the antimicrobial susceptibility to 12 commonly used antimicrobials was determined by disk diffusion. Moreover an analytical method was developed for the determination of the nine most commonly used antimicrobial agents in Ghana by using solid-phase extraction in combination with HPLC-MS/MS using electron spray ionization. The highest frequency of resistance to CoNS was observed for penicillin V (98%), trimethoprim (67%), and tetracycline (63%). S. haemolyticus was the most common isolate (75%), followed by S. epidermidis (13%) and S. hominis (6%). S. haemolyticus was also the species displaying the highest resistance prevalence (82%). 69% of the isolated CoNS were multiple drug resistant (≧ 4 antibiotics) and 45% of the CoNS were methicillin resistant. Antimicrobial agents were detected in 64% of the analysed urine samples (n = 121) where the most frequently detected antimicrobials were ciprofloxacin (30%), trimethoprim (27%), and metronidazole (17%). The major findings of this study was that the prevalence of detected antimicrobials in urine was more frequent than the use reported by the patients and the prevalence of resistant S. haemolyticus was more frequent than other resistant CoNS species when antimicrobial agents were detected in the urine.

Phase I, open-label, safety and pharmacokinetic study to assess bronchopulmonary disposition of intravenous eravacycline in healthy men and women.

This study evaluated the pulmonary disposition of eravacycline in 20 healthy adult volunteers receiving 1.0 mg of eravacycline/kg intravenously every 12 h for a total of seven doses over 4 days. Plasma samples were collected at 0, 1, 2, 4, 6, and 12 h on day 4, with each subject randomized to undergo a single bronchoalveolar lavage (BAL) at 2, 4, 6, or 12 h. Drug concentrations in plasma, BAL fluid, and alveolar macrophages (AM) were determined by liquid chromatography-tandem mass spectrometry, and the urea correction method was used to calculate epithelial lining fluid (ELF) concentrations. Pharmacokinetic parameters were estimated by noncompartmental methods. Penetration for ELF and AM was calculated by using a ratio of the area under the concentration time curve (AUC0-12) for each respective parameter against free drug AUC (fAUC0-12) in plasma. The total AUC0-12 in plasma was 4.56±0.94 µg·h/ml with a mean fAUC0-12 of 0.77±0.14 µg·h/ml. The eravacycline concentrations in ELF and AM at 2, 4, 6, and 12 h were means±the standard deviations (µg/ml) of 0.70±0.30, 0.57±0.20, 0.34±0.16, and 0.25±0.13 with a penetration ratio of 6.44 and 8.25±4.55, 5.15±1.25, 1.77±0.64, and 1.42±1.45 with a penetration ratio of 51.63, respectively. The eravacycline concentrations in the ELF and AM achieved greater levels than plasma by 6- and 50-fold, respectively, supporting further study of eravacycline for patients with respiratory infections.

One-pot synthesis of high fluorescent carbon nanoparticles and their applications as probes for detection of tetracyclines.

Herein, a novel strategy for synthesizing fluorescent carbon nanoparticles (CPs) with a quantum yield of approximately 7.1% has been well established by mixing l-cysteine, diphosphorus pentoxide and water. Compared with other current protocols, the method described here displayed various advantages including friendly manipulations, low cost, and rapid reactions. Subsequently, we applied the CPs prepared here for detections of tetracyclines (TCs). Briefly, the fluorescence intensity of CPs was quenched once TCs were introduced. Based on this phenomenon, TCs were analyzed respectively accompanyed with satisfactory detection limits and linear ranges. Significantly, the practicability of this sensing method was further validated by assaying TC in human urine samples and pharmaceutical preparations, confirming its potential to broaden avenues for detecting TCs. Additionally, the CPs could serve as fluorescent powder and ink followed by a simple post-treatment, suggesting their promising applications.

Post-run target screening strategy for ultra high performance liquid chromatography coupled to Orbitrap based veterinary drug residue analysis in animal urine.

The performance of liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) post-run target screening for veterinary drug residue analysis (sulfonamides, tetracyclines and quinolones) in animal urine has been critically evaluated. It was found that retention time information still remains an essential information and that accurate masses together with relative isotopic abundance data alone are not sufficient for many residue applications. Post-run target screening requires the careful setting of parameters to achieve near zero false negative (above a defined threshold level) and a manageable numbers of false positive findings. HRMS offers many possibilities for the reduction of false positives (e.g. isotopic ratio, isotopic fine structure, exact mass of fragment ions). However, the successful use of such tools requires a sufficient ion intensity. This is often not available when trace level compounds are to be detected. Nevertheless, the proposed method is sufficiently sensitive to detect the veterinary drugs at the relevant concentration levels in urine. This means that the approach is well suited to significantly reduce the number of corresponding meat samples which have to be analyzed in a final step for the regulatory relevant quantification of residue levels in meat. The semi-quantitative screening of many samples for a large number of analytes within a short period of time requires the availability of software tools which provide fast and reliable answers.

Improved nonaqueous capillary electrophoresis for tetracyclines at subparts per billion level.

A NACE method with laser-induced fluorescence detection was modified for sensitive detection of 4 tetracyclines (TCs) in biological samples and feeds. The changes in injection mode, injection times, id of capillary, excitation wavelength, and the use of surfactant and sample stacking technique all contributed to improved LODs of TCs to sub-ng/mL level. With the optimized conditions, the instrumental LODs could reach 1.33 ng/mL for chlorotetracycline (CTC) and 13.3 ng/mL for TC, oxytetracycline (OTC), and doxycycline (DC), an improvement of 10-100-fold over past studies. A simple SPE procedure was further developed for the extraction and concentration of TCs in plasma, urine, feed, and milk. Taken together, the instrumental LOD and feasible SPE concentration factors the overall LODs for CTC could reach 65 pg/mL in feed and milk and 260 pg/mL in plasma and urine. Detection limits for TC, OTC, and DC at sub-ng/mL level were also achieved. The modified CE-LIF method was found to be less complicated and more sensitive than the best current methods using UV or LIF detection, and has been applied successfully to assess oral absorption of DC in swine and chickens and to confirm suspected TC-positive bovine serum samples.

Analysis of tetracyclines in raw urine by column-switching high-performance liquid chromatography and tandem mass spectrometry.

The aim of the project was to develop a fast and reliable method for the quantification of the three tetracyclines: tetracycline, oxytetracycline and chlortetracycline in urine. The method is based on column-switching high-performance liquid chromatography with detection by MS-MS. Buffer is added to the sample before it is injected into the chromatographic system, and the first column which is an internal surface reversed-phase column separates the tetracyclines from the bulk of other compounds in urine. The tetracyclines are collected and concentrated on the analytical column before they are separated and eluted into the mass spectrometer in which the tetracycline are detected. The mass spectrometer is a triple quadrupole instrument and is equipped with an electrospray ion source. The MH+ ions are selected in the first quadrupole and collisionally activated in the collision cell. Upon collision, activation all three tetracyclines form fragment ions which could be assigned as: [M+H-H2O-NH3]+ which are selected in the sond mass filter. The detection limits for all three tetracyclines are about 10 ppb, and the calibration curves are linear from 10 to 1000 ppb.

Spectrofluorimetric determination of tetracycline and anhydrotetracycline in serum and urine.

A spectrofluorimetric method, involving alkaline degradation and formation of a magnesium complex, is described for the determination of tetracycline (TC) and anhydrotetracycline (ATC) in their mixed solution. Tetracycline is degraded and determined in alkaline solution. This treatment of ATC produces almost no fluorescence, but a fluorescent magnesium complex forms at pH 7.5. Several synthetic samples of TC and ATC, with TC:ATC ratios ranging from 50:1 to 1:50, were analysed. The recoveries of TC and ATC are about 71-76 and 61-63% in serum, respectively, and are all about 100% in urine.

[Detection of antibiotics in biological materials for purposes of toxicologic analysis. II. Detection of tetracyclines in biological materials]. / Prukaz nekterých antibiotik v biologickém materiálu pro úcely toxikologické analýzy. II. Prukaz tetracyklinu v biologickém materiálu.

A method of demonstration of tetracycline antibiotics elaborated as a model was used in toxicological practice. Urines of patients were analyzed after therapeutic doses and after large doses taken with suicidal intentions. At the same time, also excretion of these antibiotics from the organism was investigated. All antibiotics under study were found in the original form; in tetracycline other substances, identified as 4-epitetracycline and 4-epianhydrotetracycline, regularly appeared. A question is discussed whether these substances can be considered to be real metabolites or only products of epimerization. The importance of 4-epianhydrotetracycline for analytical purpose and interpretation of the finding is pointed out.

Liquid chromatographic and flow injection analysis of tetracycline using sensitized europium (III) luminescence detection.

Europium (III) can be used as a luminescent chromophore for detection in the liquid chromatographic and flow injection analysis of tetracycline. Detection is dependent upon an intramolecular energy transfer from the tetracycline to Eu (III). In liquid chromatography, the Eu (III) is added post-column as a complex with ethylenediaminetetraacetic acid. The post-column phase also serves to adjust the pH for optimum sensitivity. The method is highly selective for tetracycline since few compounds are capable of transferring energy to Eu (III). Fluorescent impurities that would otherwise interfere in flow injection analysis can be eliminated through the use of a delay time between the source pulse and the start of data acquisition. The detection limits for tetracycline using sensitized Eu (III) luminescence are better than those obtained using ultraviolet detection. The method is applied to the analysis of tetracycline in urine, blood serum, and gingival crevice fluid.