Aptamer-free upconversion nanoparticle/silk biosensor system for low-cost and highly sensitive detection of antibiotic residues.

The detection of antibiotics is crucial for safeguarding the environment, ensuring food safety, and promoting human health. However, developing a rapid, convenient, low-cost, and sensitive method for antibiotic detection presents significant challenges. Herein, an aptamer-free biosensor was successfully constructed using upconversion nanoparticles (UCNPs) coated with silk fibroin (SF), based on Förster resonance energy transfer (FRET) and the charge-transfer effect, for detecting roxithromycin (RXM). A synergistic FRET efficiency was achieved by utilizing alizarin red and RXM complexes as energy acceptors, with UCNP as the energy donor, and immobilizing an ultrathin SF protein corona within 10 nm. The biosensor detects RXM in deionized water with high sensitivity primarily through monolayer adsorption, with a detection range of 1.0 nM-141.6 nM and a detection limit as low as 0.68 nM. The performance of this biosensor was compared with the ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for detecting antibiotics in river water separately and a strong correlation between the two methods was observed. The biosensor exhibited long-term stability in aqueous solutions (up to 60 d) with no attenuation of fluorescence intensity. Furthermore, the biosensor's applicability extended to the highly sensitive detection of other antibiotics, such as azithromycin. This study introduces a low-cost, eco-friendly, and highly sensitive method for antibiotic detection, with broad potential for future applications in environmental, healthcare, and food-related fields.

Combined toxicity of erythromycin and roxithromycin and their removal by Chlorella pyrenoidosa.

The ecological effects of antibiotics in surface water have attracted increasing research attention. In this study, we investigated the combined ecotoxicity of erythromycin (ERY) and roxithromycin (ROX) on the microalgae, Chlorella pyrenoidosa, and the removal of ERY and ROX during the exposure. The calculated 96-h median effect concentration (EC50) values of ERY, ROX, and their mixture (2:1 w/w) were 7.37, 3.54, and 7.91 mg∙L-1, respectively. However, the predicted EC50 values of ERY+ROX mixture were 5.42 and 1.51 mg∙L-1, based on the concentration addition and independent action models, respectively. This demonstrated the combined toxicity of ERY+ ROX mixture showed an antagonistic effect on Chlorella pyrenoidosa. During the 14-d culture, low-concentration (EC10) treatments with ERY, ROX, and their mixture caused the growth inhibition rate to decrease during the first 12 d and increase slightly at 14 d. In contrast, high-concentration (EC50) treatments significantly inhibited microalgae growth (p < 0.05). Changes in the total chlorophyll contents, SOD and CAT activities, and MDA contents of microalgae suggested that individual treatments with ERY and ROX induced higher oxidative stress than combined treatments. After the 14-d culture time, residual Ery in low and high concentration Ery treatments were 17.75% and 74.43%, and the residual Rox were 76.54% and 87.99%, but the residuals were 8.03% and 73.53% in ERY+ ROX combined treatment. These indicated that antibiotic removal efficiency was higher in combined treatments than that in individual treatments, especially at low concentrations (EC10). Correlation analysis suggested that there was a significant negative correlation between the antibiotic removal efficiency of C. pyrenoidosa and their SOD activity and MDA content, and the enhanced antibiotic removal ability of microalgae benefited from increased cell growth and chlorophyll content. Findings in this study contribute to predicting ecological risk of coexisting antibiotics in aquatic environment, and to improving biological treatment technology of antibiotics in wastewater.

Occurrence and distribution of antibiotics in coastal water of the Taizhou Bay, China: impacts of industrial activities and marine aquaculture.

The occurrence, spatial distribution, and source analysis of antibiotics in global coastal waters and estuaries are not well documented or understood. Therefore, the distribution of 14 antibiotics in inflowing river and bay water of Taizhou Bay, East China Sea, was studied. Thirteen antibiotics, excluding roxithromycin (ROM), were all detected in inflowing river and bay water. The total antibiotic concentrations in bay water ranged from 3126.62 to 26,531.48 ng/L, which were significantly higher than those in the inflowing river (17.20-25,090.25 ng/L). Macrolides (MAs) and sulfonamides (SAs) were dominant in inflowing river (accounting for 24.40% and 74.9% of the total antibiotic concentrations, respectively), while SAs in bay water (93.6% of the total concentrations). Among them, clindamycin (CLI) (concentration range: ND-8414 ng/L, mean 1437.59 ng/L) and sulfadimidine (SMX) (ND-25,184.00 ng/L, mean concentrations: 9107.88 ng/L) were the highest in those surface water samples. Source analysis showed that MAs and SAs in the inflowing river mainly came from the wastewater discharge of the surrounding residents and pharmaceutical companies, while SAs in the bay water mainly came from surrounding industrial activities and mariculture. However, the contribution of the inflowing river to the bay water cannot be ignored. The risk assessment showed that SMX and ofloxacin (OFX) have potential ecological risks. These data will support the various sectors of the environment in developing management strategies and to prevent antibiotic pollution.

Chemoinformatic Screening for the Selection of Potential Senolytic Compounds from Natural Products.

Cellular senescence is a cellular condition that involves significant changes in gene expression and the arrest of cell proliferation. Recently, it has been suggested in experimental models that the elimination of senescent cells with pharmacological methods delays, prevents, and improves multiple adverse outcomes related to age. In this sense, the so-called senoylitic compounds are a class of drugs that selectively eliminates senescent cells (SCs) and that could be used in order to delay such adverse outcomes. Interestingly, the first senolytic drug (navitoclax) was discovered by using chemoinformatic and network analyses. Thus, in the present study, we searched for novel senolytic compounds through the use of chemoinformatic tools (fingerprinting and network pharmacology) over different chemical databases (InflamNat and BIOFACQUIM) coming from natural products (NPs) that have proven to be quite remarkable for drug development. As a result of screening, we obtained three molecules (hinokitiol, preussomerin C, and tanshinone I) that could be considered senolytic compound candidates since they share similarities in structure with senolytic leads (tunicamycin, ginsenoside Rb1, ABT 737, rapamycin, navitoclax, timosaponin A-III, digoxin, roxithromycin, and azithromycin) and targets involved in senescence pathways with potential use in the treatment of age-related diseases.

Correlated quantification using microbiological and electrochemical assays for roxithromycin determination in biological and pharmaceutical samples.

Microbiological and electrochemical assays, applying the cylinder-plate and differential pulse voltammetry as techniques, are reported for the quantitative determination of roxithromycin in serum and solid pharmaceutical form. The microbiological assay is based upon the inhibitory effect of this drug on the strain Bacillus subtilis ATCC 9372 used as the test microorganism. Linearity of the calibration curve was observed over the concentration range of 8.37-83.70 µg mL-1, with relative standard deviation values less than 5.0%. The electrochemical behavior of roxithromycin was studied at a graphite screen-printed electrode modified with graphene by using cyclic voltammetry and differential pulse voltammetry. The current value of the oxidative peak obtained for roxithromycin at 0.65 V vs. Ag/AgCl in 0.03 mol L-1 phosphate buffer solution (pH 7.0) with a scan rate of 0.1 V-1 is a linear function of the concentration in a range of 4.19-83.70 µg mL-1 (5-100 µmol L-1). A comparative study was carried out and both methods were applied for the determination of roxithromycin in solid dosage forms and spiked serum. The bioassay results of human serum samples were in accordance with the electrochemical ones (R2 = 0.988, P < 0.001), and the Bland-Altman method also showed good agreement between the values obtained by both procedures. Moreover, the statistical comparison indicated that there was no significant difference between the proposed techniques regarding both accuracy and precision.

Interactive effects of roxithromycin and freshwater microalgae, Chlorella pyrenoidosa: Toxicity and removal mechanism.

Roxithromycin (ROX) has received increasing concern due to its large usage, ubiquitous detection in environment and high ecotoxicology risk. This study investigated the acute and chronic effects of ROX on the growth, chlorophyll, antioxidant enzymes, and malonaldehyde (MDA) content of Chlorella pyrenoidosa, as well as the removal mechanism of ROX during microalgae cultivation. The calculated 96 h median effective concentration of ROX on yield (EyC50) and specific growth rate (ErC50) of C. pyrenoidosa was 0.81 and 2.87 mg/L, respectively. After 96 h exposure, 1.0 ~ 2.0 mg/L of ROX significantly inhibited the synthesis of chlorophyll and promoted the activities of SOD and CAT (p < 0.05). The MDA content increased with the ROX concentration increasing from 0.5 ~ 1.0 mg/L, and then decreased to 105.76% of the control exposure to 2.0 mg/L ROX, demonstrating the oxidative damage could be moderated by the upregulation of SOD and CAT activities. During the 21 d chronic exposure, low concentration of ROX (0.1 and 0.25 mg/L) showed no significant effect on the growth and chlorophyll content of algae during the first 14 d, but significantly inhibited the growth of algae and the synthesis of chlorophyll at 21 d (p < 0.05 or p < 0.01). 1.0 mg/L ROX significantly inhibited the growth of microalgae during 3 ~ 21 d and the synthesis of chlorophyll at 7 ~ 21 d. High concentration and long-term exposure of low concentration of ROX caused the SOD and CAT activities and MDA content to increase, demonstrating a higher level of oxidative damage of microalgae. During the first 14 d, abiotic removal of ROX played a more important role, contributing about 12.21% ~ 21.37% of ROX removal. After 14 d, the biodegradation of ROX by C. pyrenoidosa gradually became a more important removal mechanism, contributing about 45.99% ~ 53.30% of ROX removal at 21 d. Bio-adsorption and bioaccumulation both played minor roles in the removal of ROX during algae cultivation.

Occurrence and ecological risk of pharmaceutical and personal care products in surface water of the Dongting Lake, China-during rainstorm period.

The pharmaceutical and personal care product (PPCP) residues in freshwater lakes are being highlighted around the world. The occurrence and ecological risk of 34 PPCPs classified as antibiotics, non-steroidal anti-inflammatory drugs (NSAID), cardiovascular drugs, psychotropic drugs, anti-inflammatory drugs, psychostimulants, and pesticides during rainstorm period in surface water of the Dongting Lake, China, were studied. Twenty-six out of thirty-four PPCPs were detected, and the total concentrations of antibiotics ranged from 0.15 to 214.75 ng L-1 in surface water. The highest average concentration was observed for diclofenac, followed by diethyltoluamide (DEET). The PPCP concentrations were much lower in Dongting Lake compared to other rivers and lakes due to the strong dilution effect of rainstorm, while the detection rate remains high. Caffeine and DEET were detected with 100% frequency in Dongting Lake, and the detection rates of diclofenac, mefenamic acid, and roxithromycin were above 90%. The pollution levels of antibiotics decreased in the order of East Dongting Lake > South Dongting Lake > West Dongting Lake, which may be related to the distribution of aquaculture plants, sewage treatment plants, and population density. The risk quotient (RQ) method was used to evaluate ecological environment risk under the worst case and the results suggested that clarithromycin, diclofenac, roxithromycin, and erythromycin might pose a significant risk to aquatic organisms in Dongting Lake, especially clarithromycin. This study can provide data support for further research on the dilutive effect and mechanism of rainwater runoff on PPCPs in lakes on a large scale.

Single and combined effects of microplastics and roxithromycin on Daphnia magna.

There is a rising concern about the pollution of microplastics (plastic particles < 5 mm) in water due to their physicochemical properties, especially their interaction with organic contaminants; however, such knowledge is still limited. The mass production and consumption of medication for the treatment of infectious diseases in human and animals have led to the ubiquity of antibiotics in the environment. We studied the single and joint effects of microplastics (1-µm and 10-µm polystyrene particles, PS) and roxithromycin (ROX) on Daphnia magna through the acute and sublethal toxicity tests. The 48-h median effective concentration (EC50) of 1-µm and 10-µm PS to D. magna was 66.97 mg/L and 199.94 mg/L, respectively, while the value of ROX was 20.28 mg/L. Malondialdehyde (MDA) levels and the activities of four enzymatic biomarkers, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), were further detected to assess the oxidative stress caused in D. magna caused. The results showed that 48-h exposure to PS (0.1 mg/L) or ROX (0.01 mg/L) alone activated the activities of CAT and GST and MDA levels. When compared with the ROX alone, the responses of GPx and MDA in D. magna co-exposed to 1-µm PS were significantly decreased, while co-exposure to 10-µm PS significantly decreased the responses of GST and MDA. Furthermore, the integrated biomarker response version 2 (IBRv2) analysis revealed that co-exposure to 1-µm PS and ROX led to the strongest biological responses in D. magna. Our findings underlined that microplastics should be a concern when they interact with the co-existence of pollutants in the aquatic environment.

Degradation of typical macrolide antibiotic roxithromycin by hydroxyl radical: kinetics, products, and toxicity assessment.

The degradation of roxithromycin (ROX) by hydroxyl radical (·OH) generated by UV/H2O2 was systematically investigated in terms of degradation kinetics, effects of water chemistry parameters, oxidation products, as well as toxicity evaluation. The degradation of ROX by UV/H2O2 with varying light irradiation intensity, initial ROX concentration, and H2O2 concentration in pure water and wastewater all followed pseudo-first-order kinetics. The second-order rate constant for reaction between ROX and ·OH is 5.68 ± 0.34 × 109/M/s. The degradation rate of ROX increased with the pH; for instance, the apparent degradation rates were 0.0162 and 0.0309/min for pH 4 and pH 9, respectively. The presence of natural organic matter (NOM) at its concentrations up to 10 mg C/L did not significantly affect the removal of ROX. NO3- and NO2- anions inhibited the degradation of ROX due to the consumption of ·OH in reactions with these ions. Fe3+, Cu2+, and Mg2+ cations inhibited the degradation of ROX, probably because of the formation of ROX-metal chelates. A total of ten degradation products were tentatively identified by HPLC/LTQ-Orbitrap XL MS, which mainly derived from the attack on the oxygen linking the lactone ring and the cladinose moiety, tertiary amine and oxime side chain moiety by ·OH. The toxicity evaluation revealed that UV/H2O2 treatment of ROX induced the toxicity to bioluminescent bacteria increased.

Screening of antimicrobials in animal-derived foods with desorption corona beam ionization (DCBI) mass spectrometry.

The relatively new technique, desorption corona beam ionization (DCBI), was coupled with an ion-trap mass spectrometer for the rapid detection of 10 antimicrobials in animal-derived food. Under positive ion mode, 10 common antimicrobials were identified without prior sample preparation. With simple pre-treatment, semi-quantification based on peak area was achieved. There was a good correlation (R2 > 0.99) in the rational linear ranges (1-3 orders of magnitude) for all 10 target analytes. Compared with a conventional liquid chromatographic method, the DCBI-MS method was highly sensitive; e.g., roxithromycin was detected at 0.02 µg/g, whereas the value was 0.2 mg/kg in the European Union Commission Regulation (No 37/2010) and 0.3 µg/mL in a recently reported LC method. The proposed method allowed for the simple, rapid, sensitive and specific detection of antimicrobials in animal-derived foods. Our approach could be used for other atmospheric pressure chemical ionization (APCI) related ambient mass spectrometry methods to detect other compounds.

Stabilization Mechanism of Roxithromycin Tablets Under Gastric pH Conditions.

Macrolide antibiotics are widely used at clinical sites. Clarithromycin (CAM), a 14-membered macrolide antibiotic, was reported to gelate under acidic conditions. Gelation allows oral administration of acid-sensitive CAM without enteric coating by hindering the penetration of gastric fluid into CAM tablets. However, it is unknown whether this phenomenon occurs in other macrolide antibiotics. In this study, we examined the gelation ability of 3 widely used macrolide antibiotics, roxithromycin (RXM), erythromycin A, and azithromycin. The results indicated that not only CAM but also RXM gelated under acidic conditions. Erythromycin A and azithromycin did not gelate under the same conditions. Gelation of RXM delayed the disintegration of the tablet and release of RXM from the tablet. Disintegration and release were also delayed in commercial RXM tablets containing disintegrants. This study showed that 2 of the 4 macrolides gelated, which affects tablet disintegration and dissolution and suggests that this phenomenon might also occur in other macrolides.

Optimization of Robust HPLC Method for Quantitation of Ambroxol Hydrochloride and Roxithromycin Using a DoE Approach.

The aim of this work was to develop and optimize a robust HPLC method for the separation and quantitation of ambroxol hydrochloride and roxithromycin utilizing Design of Experiment (DoE) approach. The Plackett-Burman design was used to assess the impact of independent variables (concentration of organic phase, mobile phase pH, flow rate and column temperature) on peak resolution, USP tailing and number of plates. A central composite design was utilized to evaluate the main, interaction, and quadratic effects of independent variables on the selected dependent variables. The optimized HPLC method was validated based on ICH Q2R1 guideline and was used to separate and quantify ambroxol hydrochloride and roxithromycin in tablet formulations. The findings showed that DoE approach could be effectively applied to optimize a robust HPLC method for quantification of ambroxol hydrochloride and roxithromycin in tablet formulations. Statistical comparison between results of proposed and reported HPLC method revealed no significant difference; indicating the ability of proposed HPLC method for analysis of ambroxol hydrochloride and roxithromycin in pharmaceutical formulations.

A novel RP-HPLC method for the detection and quantification of roxithromycin in topical delivery studies.

A novel HPLC method with UV detection for the identification and quantification of roxithromycin (ROX) during in vitro skin penetration studies has been developed and validated. The method proved to be simple and rapid with isocratic elution (flow rate: 1.0 mL/min) of ROX, using a C18 column and UV detection at 205 nm. The mobile phase consisted of 0.06 M potassium di-hydrogen orthophosphate buffer (pH adjusted to 7.4 with sodium hydroxide) and acetonitrile in a 50:50 (v/v) ratio. This method showed linearity across the concentration range of 5 - 1000 µg/mL with a correlation coefficient of 0.9999. An average recovery of 101.78% was obtained. Limit of detection (LOD) and lower limit of quantification (LLOQ) values proved that ROX can still be detected at a concentration level of 0.3 µg/mL and accurately quantified at a concentration of 0.5 µg/mL. The specificity testing during method validation proved that this method is suitable for the accurate detection and quantification of ROX even when combined with different compounds typically used during the formulation of topical delivery systems.

Interactive effects of selected pharmaceutical mixtures on bioaccumulation and biochemical status in crucian carp (Carassius auratus).

The aim of this study was to evaluate the interactive effects of fluoxetine (FLU), roxithromycin (ROX) and propranolol (PRP) on the bioaccumulation and biochemical responses in the crucian carp Carassius auratus. After 7 days of binary exposure (ROX + FLU and PRP + FLU), the addition of waterborne FLU at nominal concentrations of 4, 20 and 100 µg L(-1) significantly increased the accumulation of ROX and PRP in fish livers in most cases, although elevated ROX and PRP bioaccumulation levels were not observed in muscles or gills. The inductive response of 7-ethoxyresorufin O-deethylase (EROD) to PRP and that of 7-benzyloxy-4-trifluoromethyl-coumarin O-dibenzyloxylase (BFCOD) to ROX were inhibited by the co-administration of FLU at all tested concentrations. Correspondingly, marked inhibition of CYP1A and CYP3A mRNA expression levels was observed in the livers of fish co-treated with FLU + PRP and FLU + ROX relative to their PRP- and ROX-only counterparts, respectively. In addition, as reflected by superoxide dismutase (SOD) activity and malondialdehyde (MDA) content, co-exposure to ROX + FLU and PRP + FLU seemed to induce stronger antioxidant responses than single pharmaceutical exposure in fish livers. This work indicated that the interactive effects of pharmaceutical mixtures could lead to perturbations in the bioaccumulation and biochemical responses in fish.

Different amorphous solid-state forms of roxithromycin: A thermodynamic and morphological study.

The striking impact that different preparation methods have on the characteristics of amorphous solid-state forms has attracted considerable attention during the last two decades. The pursuit of more extensive knowledge regarding polyamorphism therefore continues. The aim of this study was firstly, to investigate the influence of different preparation techniques to obtain amorphous solid-state forms for the same active pharmaceutical ingredient, namely roxithromycin. The preparation techniques also report on a method utilizing hot air, which although it is based on a melt intermediary step, is considered a novel preparation method. Secondly, to conduct an in-depth investigation into any physico-chemical differences between the resulting amorphous forms and thirdly, to bring our findings into context with that of previous work done, whilst simultaneously discussing a well-defined interpretation for the term polyamorphism and propose a discernment between true polyamorphism and pseudo-polyamorphism/atypical-polyamorphism. The preparation techniques included melt, solution, and a combination of solution-mechanical disruption as intermediary steps. The resulting amorphous forms were investigated using differential scanning calorimetry, X-ray powder diffraction, hot-stage microscopy, scanning electron microscopy, and vapor sorption. Clear and significant thermodynamic differences were determined between the four amorphous forms. It was also deduced from this study that different preparation techniques have a mentionable impact on the morphological properties of the resulting amorphous roxithromycin powders. Thermodynamic properties as well as the physical characteristics of the amorphous forms greatly governed other physico-chemical properties i.e. solubility and dissolution.

Roxithromycin potency quantification in pharmaceutical preparation by applying a validated bioassay method and comparison with HPLC analysis.

Roxithromycin (RXM) is used to treat bacterial infections. An alternative bioassay for the assessment of the potency of this drug in pharmaceutical formulations has not been reported earlier. This study reports the development and validation of cost-effective, simple, sensitive, precise, accurate and reproducible one-level agar diffusion (5+1) bioassay for estimation of potency and bioactivity of RXM in tablet. Among six tested microbial strains, Streptococcus pneumoniae (MTCC-1935) was used as the most susceptible strain against RXM. Bioassay was optimized by investigating buffer pH, inoculums and reference standard concentration. The results of proposed bioassay displayed high linearity, precision, accuracy, robustness and specificity. All potency results were statistically analyzed and found to be linear (R(2)=0.9957) in between 2.0 and 6.0µg/mL, precise with relative standard deviation (RSD) of repeatability intra-assay below 1.5%, and intermediate precision between day RSD 0.39% and accurate (100.68%). The bioassay and previously validated HPLC methods were compared, which indicated that there was no significant difference between these two methods. The results demonstrated the validity of the proposed microbial assay, which allows reliable quantitation of RXM in pharmaceutical samples and therefore can be used as a substitute alternative methodology for the routine quality control of this medicine, in situation when HPLC is not affordable in the laboratory.

Evaluation of the potential for trophic transfer of roxithromycin along an experimental food chain.

Pharmaceuticals have been recognized as a new class of environmental pollutants in recent years. But data about their potential for transfer and biomagnification in aquatic food chains are still lacking. In this study, bioaccumulation of the macrolide antibiotic roxithromycin (ROX) was determined in an experimental aquatic food chain involving the green algae Scenedesmus obliquus, the water flea Daphnia magna and the crucian carp Carassius auratus. After 48 h of exposure, S. obliquus accumulated ROX from media, with bioconcentration factors (BCFs) of 74.6, 46.3, and 24.5 l kg(-1) at nominal exposure concentrations of 4, 20, and 100 µg l(-1), respectively. After 48 h of feeding ROX-contaminated algae, D. magna was able to accumulate ROX in all three concentration treatments, but biomagnification did not occur at this trophic level, as biomagnification factors (BMFs) varied from 0.21 to 0.29 in different concentration treatments were well below one. In tissues (muscle, gill, liver, and bile) of C. auratus fed with contaminated daphnia for 8 days, no biomagnification was observed. However, this species did accumulate a certain degree of ROX through food chain transfer, and the tissue burden was greatest in the liver > muscle > gill and lowest in the bile. This work suggests that the trophic transfer should be a matter of concern for ecological risk assessments of pharmaceutical substances in aquatic food webs.

Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes.

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO-CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100-150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC50=0.13-0.2ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC50=1.6ng/ml and LOD=0.14ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.

Continuous, quantitative monitoring of roxithromycin in human saliva by flow injection chemiluminescence analysis.

Human saliva quantitative monitoring of roxithromycin (ROX) at picomolar-level by flow injection (FI) chemiluminescence (CL) analysis is described for the first time, to our knowledge. Monitoring was based on the CL intensity from luminol-BSA reaction, which can be quenched in the presence of ROX, with the decreasing CL intensity linearly proportional to the logarithm of the ROX concentration, ranging from 0.6 to 1000 pmol·L(-1). The detection limit of the proposed method for the determination of ROX was as low as 0.2 pmol·L(-1) (3σ), and the relative standard deviations were less than 4.0% (n = 7). A complete analytical process, including sampling and washing for ROX determination, conducted at a flow rate of 2.0 mL·min(-1), was performed completely within 30 s, yielding a sample efficiency of 120 h(-1). The proposed method was successfully applied to the determination of ROX in human saliva and serum samples with recoveries from 90.9% to 110.1%. The continuous monitoring of ROX in human saliva after oral intake showed that the total elimination ratio was 87.1% during 24 h, and the pharmacokinetic parameters were 0.97 ± 0.18 h(-1) for the absorption rate constant K(a), 0.082 ± 0.010 h(-1) for the elimination rate constant K(e), and 8.56 ± 1.11 h for the elimination half-life time t(1/2). It was also found that ROX in human saliva and urine simultaneously reached the maximum at 2 h with the concentration correlate ratio of 0.97.

Liquid chromatographic determination of roxithromycin: application to stability studies.

A simple, stability-indicating, reversed-phase liquid chromatographic method has been developed for the determination of roxithromycin in the presence of its forced alkaline, oxidative and ultraviolet degradation products. Reversed-phase chromatography was conducted using an ODS C18 (150 × 4.6 mm i.d.) column at ambient temperature with ultraviolet detection at 215 nm. A mobile phase consisting of 0.03 M potassium dihydrogen phosphate buffer-methanol (40:60, v/v) adjusted to pH 4.5 was used for the separation of the studied drug and its degradation products at a flow rate of 1 mL/min. The method showed good linearity over the concentration range of 10.0-150.0 µg/mL with a detection limit of 2.5 µg/mL and quantification limit of 8.4 µg/mL. The proposed method was successfully applied for the analysis of roxithromycin in its commercial tablets; the obtained results were favorable compared with those obtained by the official method. Furthermore, content uniformity testing of the studied tablets was also conducted. The method was also utilized to investigate the kinetics of the different degradation products of the drug. The first-order rate constant, half-life time and activation energy of the degradation reactions were calculated.