New carbon dots based on glycerol and urea and its application in the determination of tetracycline in urine samples.

The current study proposes a fast one-pot microwave assisted synthesis of new carbon dots (CDs) based on glycerol and urea. The novel carbon nanoparticles (GUCDs) have been appropriately characterized and exhibited good luminescent properties with a quantum yield of about 9.8%. Interestingly, the GUCDs are able to selectively interact with tetracycline class antibiotics, which produce a decrease in the native fluorescence of the CDs. On the base of these features, a new analytical method has been developed for the determination of tetracycline. The proposed method has shown satisfactory analytical parameters, such as good linearity range -between 0.5 and 25 µM (R2 = 0.9997)- and an acceptable detection limit (165 nM). Moreover, the new method has been successfully applied for tetracycline determination in urine samples with good recoveries (94.7-103%) and precision (4.6 RSD%).

Development, validation and application of a novel HPLC-MS/MS method for the measurement of minocycline in human plasma and urine.

New treatments are urgently required to treat infections caused by multi-drug resistant Acinetobacter baumanni,. To address this need, a new formulation of Minocin®, (minocycline for injection) has been developed that allows for higher doses of minocycline to be administered. Phase 1 clinical trials were conducted in healthy volunteers to assess the safety and pharmacokinetics (PK) of this new formulation at higher doses. In order to generate PK data, novel, selective and simple HPLC-MS/MS based assays were developed and validated for the determination of minocycline (MC) in human plasma and urine. The respective working ranges were 0.05 to 30 mg/L and 0.1 to 30 mg/L. Removal of endogenous proteins with trichloroacetic acid was used as a simple means of extracting MC from the samples. An analogue, tetracycline was used as the internal standard (IS). Chromatographic separation, including that of MC from its 4-epimer (4-EMC), was achieved on a Waters XBridge BEH C18 column (50 x 4.6 mm ID, 5 µm) with gradient elution. The mobile phases comprised water containing 5 mM ammonium formate at a pH of 2.5, and methanol containing 5 mM ammonium formate. The internal standard (IS) was tetracycline, a structural analogue of minocycline. The methods were fully validated and met regulatory acceptance criteria for intra-run and inter-run accuracy and precision, carryover, dilution integrity and matrix effects. Mean extraction recoveries ranged between 64.3% and 84.6% for MC and 64.3% for the IS. There was no significant ion suppression or enhancement for MC or the IS. The validated assays were successfully applied to 1423 plasma and 689 urine samples from a Phase 1 clinical study. There was no evidence of instability, or significant interconversion between MC and 4-EMC, in stored clinical samples, spiked plasma and urine samples, or their extracts, under various test conditions.

Collision cross section (CCS) as a complementary parameter to characterize human and veterinary drugs.

In the context of human and veterinary drugs identification, ion mobility spectrometry (IMS) in combination with mass spectrometry (MS) may provide a relevant complementary piece of information to mass-to-charge ratio (m/z), the so-called collision-cross-section (CCS). Up to now, however, the application of CCS as identification parameter has not been fully investigated due to the reduced number of these drugs that have being characterized in terms of CCS. This work proposes a CCS database for 92 human and veterinary drugs, including eighteen benzimidazoles, eleven 5-nitroimidazoles, eleven aminoglycosides, nineteen quinolones, eighteen ß-lactams, ten sulfonamides and five tetracyclines. Among them, 37 drugs have been characterized in terms of CCS for the first time. The CCS values of the other 55 compounds have been compared with those from a recently published database in order to evaluate inter-laboratory reproducibility, which is crucial for the implementation of the CCS as identification parameter. CCS values were measured by traveling wave ion mobility spectrometry (TWIMS) under positive ionization conditions. Nitrogen was used as drift gas in the ion mobility cell. The proposed database covers 173 ions including [M+H]+ and [M+Na]+ species. High correlation between m/z and CCS has been observed for [M+H]+ (R2 = 0.9518, n = 91) and [M+Na]+ (R2 = 0.9135, n = 82) ions. As expected, CCS values for sodium adducts are generally greater than for protonated molecules because they exhibit higher molecular weight. However, sodium adducts of aminoglycosides, ß-lactams, and of several quinolones and benzimidazoles, were characterized as more compact ions than their related protonated molecule. In addition, this work describes the fragmentation pattern observed for the studied molecules. For the first time, the main fragment ions for most of the compounds have also been characterized in terms of CCS, involving a total of 238 ions. As proof of concept, for the application of this database to biological matrices, eleven veterinary drugs in bovine urine samples were characterized in terms of CCS, showing that this parameter was not influenced by the matrix.

An automated salting-out assisted liquid-liquid microextraction approach using 1-octylamine: On-line separation of tetracycline in urine samples followed by HPLC-UV determination.

An automated salting-out assisted liquid-liquid microextraction (SALLME) procedure based on a flow system was developed as new approach for pretreatment of complex sample matrix. In this procedure 1-octylamine was investigated as novel extractant for the SALLME. The procedure involved aspiration of the 1-octylamine and sample solution into a mixing chamber of a flow system followed by their air-bubble mixing resulting to isotropic solution formation. To provide phase separation a salting-out agent solution was added into the mixing chamber. After phase separation, the micellar 1-octylamine phase containing analyte was mixed with methanol and transported to a HPLC-UV system. To demonstrate the efficiency of the suggested approach, the automated procedure was applied for the HPLC-UV determination of tetracycline as a proof-of-concept analyte in human urine samples. Under the optimal conditions, the detector response of the analytes was linear in the concentration ranges of 0.5-20 mg L-1. The limit of detection, calculated from a blank test based on 3σ, was 0.17 mg L-1. The results demonstrate that the developed approach is highly cost-effective, simple and rapid.

Carbon dots derived from rose flowers for tetracycline sensing.

Herein, an innovative and simple method for synthesizing carbon dots (CDs) with satisfactory fluorescence has been successfully established while rose flowers served as carbon source for the first time. Meanwhile, the fluorescence (FL) mechanism of current CDs was elucidated in detail by fluorescence, UV-vis, HR-TEM, and FTIR-based analyses. Subsequently, this type of CDs was employed for detecting tetracycline (TC) on the basis of the interactions between TC and CDs, and allowed quenching their fluorescence. Moreover, the proposed analytical strategy permitted detecting TC in a linear range of 1.0×10(-8)-1.0×10(-4) mol/L with a detection limit of 3.3×10(-9) mol/L at a signal-to-noise ratio of 3. Significantly, the CDs described here were further applied for fluorescent coding, demonstrating their promising future towards various applications in analytic science.

Novel and remarkable enhanced-fluorescence system based on gold nanoclusters for detection of tetracycline.

Tetracycline and Eu(3+), while coexisting, usually appear as a complex by chelating. This complex shows low fluorescence intensity, leading to its limitation of analytical goals. Gold nanoclusters (AuNCs), emerging as novel nano-material, are attracting increasing attentions in multiple fields. Herein, gold nanoclusters first function as a fluorescence-enhanced reagent rather than a conventional fluorescent-probe, and a dramatic enhanced-fluorescence system was built based on Eu(3+)-Tetracycline complex (EuTC) by introducing gold nanoclusters. Simultaneously, three types of gold nanoclusters were employed for exploring various conditions likely affecting the system, which demonstrate that no other gold nanoclusters than DNA-templated gold nanoclusters enormously caused fluorescence-enhancement of EuTC. Moreover, this enhanced-fluorescence system permitted available detection of tetracycline (TC) in a linear range of 0.01-5 µM, with a detection limit of 4 nM at a signal-to-noise ratio of 3. Significantly, the practicality of this method for detection of TC in human urine and milk samples was validated, demonstrating its advantages of simplicity, sensitivity and low cost. Interestingly, this system described here is probably promising for kinds of applications based on its dramatically enhanced-fluorescence.

Aptamer based extraction followed by electrospray ionization-ion mobility spectrometry for analysis of tetracycline in biological fluids.

An extraction method based on aptamer sorbent followed by electrospray ionization-ion mobility spectrometry (ESI-IMS) has been developed for the analysis of tetracycline in human urine and plasma samples. The effect of extraction parameters on the extraction efficiency including washing (solvent type and volume) and elution (solvent type, volume and flow rate) were investigated. Under the optimized conditions, the linear dynamic ranges for tetracycline in urine and plasma samples were found to be 0.05-5.00 µg/mL and 0.10-5.00 µg/mL with detection limits of 0.019 and 0.037 µg/mL, respectively. The extraction efficiency was 86.5% for urine and it was 82.8% for plasma samples. The relative standard deviation was 5.9% and 6.3% for six replicate measurements of tetracycline at 1 and 2 µg/mL in urine and plasma samples, respectively.

Automated annotation and quantification of metabolites in 1H NMR data of biological origin.

In (1)H NMR metabolomic datasets, there are often over a thousand peaks per spectrum, many of which change position drastically between samples. Automatic alignment, annotation, and quantification of all the metabolites of interest in such datasets have not been feasible. In this work we propose a fully automated annotation and quantification procedure which requires annotation of metabolites only in a single spectrum. The reference database built from that single spectrum can be used for any number of (1)H NMR datasets with a similar matrix. The procedure is based on the generalized fuzzy Hough transform (GFHT) for alignment and on Principal-components analysis (PCA) for peak selection and quantification. We show that we can establish quantities of 21 metabolites in several (1)H NMR datasets and that the procedure is extendable to include any number of metabolites that can be identified in a single spectrum. The procedure speeds up the quantification of previously known metabolites and also returns a table containing the intensities and locations of all the peaks that were found and aligned but not assigned to a known metabolite. This enables both biopattern analysis of known metabolites and data mining for new potential biomarkers among the unknowns.

Pharmacology of tetracycline water medication in swine.

Medicating drinking water with tetracycline is commonly used in swine production systems to treat and prevent disease outbreaks. However, little information is known of the pharmacokinetics of this medication in water formulations. Twenty-four barrows, divided into 1 control group (of nontreated animals) and 3 equally sized treatments groups (n = 6/group), were treated with tetracycline water medication for 5 d at 125, 250, and 500 mg/L. Blood samples were collected at 0 (prestudy), 4, 8, 12, 24, 32, 48, 56, 72, 80, 96, and 104 h after exposure. Data analyses consisted of a noncompartmental pharmacokinetic analysis and statistical analysis of steady state concentrations with repeated measures ANOVA and multiple-comparison testing to determine whether plasma concentrations differed among groups. Derived pharmacokinetic parameters were consistent with previously published feed and intravenous data. Plasma tetracycline concentrations at steady state were 0, 0.33, 0.47, and 0.77 microg/mL for 0-, 125-, 250-, and 500-mg/L exposures, respectively. Treatment group steady-state plasma concentrations were significantly different from plasma concentrations in control animals (P < 0.0001); however, whereas the 125- and 250-mg/L groups were significantly different from the 500-mg/L group (P < 0.0001), their mean plasma tetracycline concentrations did not differ from one another. Furthermore, the study showed that tetracycline oral bioavailability is very small. The dose response curve also shows that concentrations of plasma tetracycline increase linearly, yet not in a 1 to 1 ratio, to the direct increase in water medication dose.

Effect of a Kampo preparation, byakkokaninjinto, on pharmacokinetics of ciprofloxacin and tetracycline.

The effect on the bioavailability of the antimicrobial agents (ciprofloxacin and tetracycline), which are well known to form chelates with cationic metals such as calcium, was evaluated in 20 healthy male volunteers according to an open, random crossover fashion using a Kampo preparation, byakkokaninjinto (TJ-34) which contains various cationic metals including calcium. Each subject received a single oral dose of tetracycline (250 mg) alone or ciprofloxacin (200 mg) alone along with a single coadministration of one pack (3 g) of the Kampo preparation, at one-week intervals. Concentrations of the drugs in plasma and urine were analyzed by HPLC. Concomitant administration of the Kampo preparation significantly decreased the peak plasma concentration (C(max)) and area under the plasma concentration-time curves (AUC), but not time to reach C(max) (T(max)), of ciprofloxacin and tetracycline. However, the decrease in bioavailability of ciprofloxacin was slight (15%) compared with that of tetracycline (30%). The Kampo preparation significantly decreased the urinary recovery of tetracycline, but not ciprofloxacin, and it had no effect on the renal clearance of either ciprofloxacin or tetracycline. These results indicate that the Kampo preparation tested in this study reduces the extent of bioavailability of ciprofloxacin and tetracycline, but not renal excretion, by decreasing the gastrointestinal absorption due to the formation of insoluble chelates with calcium. We recommend that the dose timing of the Kampo preparation should be carefully controlled to avoid therapeutic failure especially for patients receiving the treatment with tetracycline.

PARAFAC and PLS applied to spectrophotometric determination of tetracycline in pharmaceutical formulation and biological fluids.

A simple and rapid analytical procedure was proposed for determination of tetracycline in pharmaceutical formulation, urine and plasma based on chemometrics methods and spectrophotometric measurements. The calibration set was constructed with twenty solutions in concentration range 0.25-13.00 microg ml(-1) for tetracycline. The procedure was repeated at nine different pH values. Partial least squares (PLS) models were built at each pH and used to determinate a set of synthetic tetracycline solutions. The best model was obtained at pH 8.00 (PLS-PH8). Parallel factor analysis (PARAFAC) model was applied to a three-way array constructed using all the pH data sets and enabled better results. The capabilities of the method for the analysis of real samples were evaluated by determination of tetracycline in pharmaceutical formulations and biological fluids with satisfactory results.

Determination of tetracycline, chlortetracycline and oxytetracycline by flow injection with inhibitory chemiluminescence detection using copper(II) as a probe ion.

This paper reports an indirect flow-injection (FI) method for the determination of the tetracycline drugs (TCs), tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC), using copper(II) as a probe ion. The method was based on the inhibition caused by these TCs to the copper(II)-catalysed chemiluminescence (CL) reaction between luminol and H(2)O(2). The CL reaction was induced on-line and injection of the sample produced negative peaks as a result of the copper(II) complexation or displacement by the analytes. The height of the peaks was proportional to the drug concentration in the sample. The choice of the catalyst ion, the concentration of luminol, H(2)O(2) and copper(II) are discussed. The linear range was 3.6 x 10(-8)-1.0 x 10(-5), 1.1 x 10(-7)-1.0 x 10(-5) and 1.9 x 10(-7)-1.0 x 10(-5) mol/L for TC, CTC and OTC, respectively. The detection limit was 5.0 x 10(-9) mol/L for TC, 1.0 x 10(-8) mol/L for CTC and 2.0 x 10(-8) mol/L for OTC (3sigma), respectively. The method was applied to the determination of TCs in pharmaceutical preparations and human urine with recoveries in the range 95-105%.

Pharmacokinetic interaction of tetracycline with non-steroidal anti-inflammatory drugs via organic anion transporters in rats.

The present study aims to investigate the pharmacokinetic interaction between non-steroidal anti-inflammatory drugs and tetracycline in rats. Pharmacokinetic parameters were determined following an intravenous administration of tetracycline (5 mg kg(-1)) to rats in the presence and absence of naproxen or diclofenac (20 mg kg(-1)). Compared to the control (given tetracycline alone), pretreatment with naproxen or diclofenac 30 min prior to tetracycline administration significantly altered the pharmacokinetics of tetracycline. Renal clearance of tetracycline was reduced by approximately three-fold in the presence of naproxen or diclofenac. Consequently, the systemic exposures (AUC) of tetracycline in the rats pretreated with naproxen or diclofenac were significantly (p < 0.05) higher than those from the control group given tetracycline alone. Furthermore, mean terminal plasma half-life of tetracycline was enhanced by two- to five-folds under the pretreatment with naproxen or diclofenac. Those results suggest that NSAIDs such as naproxen and diclofenac are effective to alter the renal elimination and pharmacokinetic profiles of tetracycline. Therefore, concomitant use of naproxen or diclofenac with tetracycline may require close monitoring for clinical consequence of potential drug interaction.

Analysis of whole skeleton 3H-tetracycline loss as a measure of bone resorption in maturing rats.

Several modifications of the 3H-tetracycline bone labeling method for measuring whole skeleton bone resorption were tested. Under steady state conditions of whole skeleton resorptive activity, bone labeling for intervals longer than 2 weeks prior to experimentation did not significantly alter the urinary 3H-tetracycline loss curve. The utilization of nonlinear regression analysis showed that the urinary loss of 3H-tetracycline was best described by double exponential equations, indicating the loss of label from two distinct and independent exchangeable bone compartments. This conclusion was supported by the finding that soft tissues were effectively depleted of 3H-tetracycline by 24 hours after the final injection of label. Hence, it was concluded that approximately 40% of the 3H-tetracycline loss from skeletal bone is associated with a "fast" compartment which is depleted within 6 or 7 days after label loading. The size and rate of 3H-tetracycline loss from the fast compartment decreased (40%) with age such that the depletion time remained constant between 8 and 24 weeks of age in both male and female rats. The remaining 60% of 3H-tetracycline loss from a "slow" compartment which was depleted in about 70 days in young (8 week) rats. This compartment, which is believed to reflect cell-mediated resorption of calcified bone; decreased in size with age in both male (50%) and female (30%) rats. The rate of label loss from this compartment, however, remained relatively high so that the depletion time decreased (approximately 35%) between 8 and 24 weeks of age. By determining whole skeletal mass and calculating these parameters on the basis of skeletal mass, we were further able to demonstrate significantly higher resorptive activity in female than in male rats by 24 weeks of age.

Renal excretion of tetracycline is transiently decreased during short-term heat exposure.

Renal blood flow is known to be reduced during intensive external heating, which may have clinical relevance for renal drug elimination as well. The effects of heat exposure in a sauna bath on tetracycline pharmacokinetics were studied in 8 healthy volunteers in an open, randomized crossover study. The subjects were given a single oral dose of tetracycline (500 mg) both in a control session and in a sauna bathing session. The heat exposure consisted of three 10-minute stays in a sauna bath (temperature 76-87 degrees C, relative humidity 27-33%) starting 20 minutes after drug administration. The stays in the steam room were separated by two 5-minute cooling periods at 23 degrees C. Venous blood samples for determination of plasma tetracycline concentrations were taken 15 min before the drug intake, 20, 40, 60 min, and 2, 3, 4, 6, 8, and 24 h after it. The control session at room temperature (23 degrees C) followed the same sampling protocol. No statistically significant differences in tetracycline plasma concentrations, Cmax, Tmax, or AUC0-24h were seen. In addition, urine was collected (0-2 h, 2-5 h, 5-8 h, and 8-24 h) for determination of the amount of tetracycline excreted unchanged. Urinary tetracycline excretion was transiently (2-5 h after drug intake) reduced in the sauna session (P < 0.05 vs. control, Wilcoxon). The other collection periods and the total urinary excretion of tetracycline (24 h) did not differ from the control session. It is concluded that urinary tetracycline excretion was transiently decreased during short-term heat exposure, but otherwise the effects of external heating on tetracycline pharmacokinetics were negligible.

Effect of bisphosphonates on the increase in bone resorption induced by a low calcium diet.

This study investigates whether bisphosphonate-treated rats are still able to adapt to low calcium supply through an increase in bone resorption assessed by measuring the urinary excretion of [3H]-tetracycline from chronically prelabeled rats. First it was shown that in this model, parathyroid hormone was responsible for the increase in bone resorption on the low calcium diet. In the second part, animals were treated with the three bisphosphonates-clodronate, alendronate, and ibandronate-given in two doses. Animals receiving a dose that already strongly inhibits bone resorption were still able to respond to a low calcium diet by increasing bone resorption, showing the potency of the latter as a stimulator of bone resorption. Higher doses were, however, able to blunt this response. As soon as the treatment was discontinued, this increase in bone resorption resumed with clodronate but not with alendronate or ibandronate.

The food-induced stimulation of bone resorption in the rat, assessed by the urinary [3H]-tetracycline excretion, is mediated by parathyroid hormone.

Using the urinary excretion of [3H]-tetracycline from prelabeled rats to monitor bone resorption, we have previously shown that food intake is associated with a rapid and large increase in bone resorption. This increase is blunted when the daily intake is fractionated into 4 portions instead of being given at once. Food fractionation also leads to a large increase in bone mass. In order to establish whether the thyroparathyroid gland is involved in this effect, thyroparathyroidectomized (TPTX) rats were studied. The food-induced increase in bone resorption was absent in TPTX rats. Therefore, the pattern of parathyroid hormone (PTH) was investigated during the development of the food-induced bone resorption, and under food fractionation. Rats were trained to eat their daily high Ca food in less than two hours. Thereafter they were given food portions of 5 or 20 grams, respectively. PTH in serum was measured at 0, 1, 2, 3, and 4 hours after food intake. In rats given the large food portions, a conspicuous increase of PTH was found. In contrast, serum PTH of rats fed the small food portion did only change to an insignificant extent. This study in rats shows that the ingestion of a large meal induces a transient increase of PTH. The present results can therefore explain the formerly observed acute increase in bone resorption following food intake and its blunting by food fractionation. It is not known, whether such a mechanism occurs also in humans. If so, these results would provide the rational basis to decrease bone resorption by food fractionation.

Evaluation of urinary pyridinium crosslink excretion as a marker of bone resorption in the rat.

The aim of this study was to evaluate the value of the urinary excretion of the pyridinium crosslinks, pyridinoline (Pyr) and deoxypyridinoline (D-Pyr), as markers of bone resorption in the rat. The excretion of the crosslinks was compared with that of urinary [3H]tetracycline ([3H]TC) excretion from chronically [3H]TC-prelabeled animals, a technique established to monitor bone resorption in the rat. Bone resorption was modulated by Ca restriction, infusion of PTH, thyroparathyroidectomy, and administration of different bisphosphonates. Furthermore, the urinary crosslinks were assessed in three different osteopetrotic mutations in the rat. We found a delayed response of Pyr and D-Pyr excretion to acute changes in bone resorption compared with [3H]TC excretion. This delay was 1 day after Ca restriction and longer after other treatments, such as PTH administration or bisphosphonate treatment, with which it was more than 3 weeks. In contrast, chronic states with stimulation or inhibition of bone resorption showed similar changes in excretion of the urinary crosslinks and [3H]TC, except after PTH administration. The excretion of the crosslinks was greatly reduced in osteopetrotic rats (op/op, tl/tl, and ia/ia) and increased to normal levels in tl/tl rats after stimulation of bone resorption by M-CSF administration. These results suggest that, in rats, urinary excretion of the pyridinium crosslinks reflects bone resorption in chronic but not always in acute conditions. The cause of this discrepancy is still unclear.

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 toxicological analysis. I. Methodologic study for the detection of tetracycline]. / Prukaz nekterých antibiotik v biologickém materiálu pro úcely toxikologické analýzy. I. Metodická studie pro prukaz tetracyklinu.

A method of the identification of tetracycline antibiotics in urine and gastric content, suitable for the routine toxicological analysis, was worked out. For the isolation, the extraction with ethyl acetate is recommended. The TLC method on Silufol layers impregnated with an aqueous solution of the disodium salt of ethylenediaminetetraacetic acid (Na2EDTA), in the concentration of 0.1 mol/l and pH value of 5 or 7.4 is used for the identification. The mobile phase chloroform--methanol--Na2EDTA 55:30:5 is suitable for the development of these layers; UV light of the wavelength 254 or 366 nm and Fast Blue B reagent are used for the detection. The conditions of the TLC (various modifications of the layer, mobile phase and detection) and the extraction solvents suitable for the isolation are discussed.