A Comprehensive Study to Determine the Residual Elimination Pattern of Major Metabolites of Amoxicillin-Sulbactam Hybrid Molecules in Rats by UPLC-MS/MS.

Amoxicillin and sulbactam are widely used in animal food compounding. Amoxicillin-sulbactam hybrid molecules are bicester compounds made by linking amoxicillin and sulbactam with methylene groups and have good application prospects. However, the residual elimination pattern of these hybrid molecules in animals needs to be explored. In the present study, the amoxicillin-sulbactam hybrid molecule (AS group) and a mixture of amoxicillin and sulbactam (mixture group) were administered to rats by gavage, and the levels of the major metabolites of amoxicillin, amoxicilloic acid, amoxicillin diketopiperazine, and sulbactam were determined by UPLC-MS/MS. The residue elimination patterns of the major metabolites in the liver, kidney, urine, and feces of rats in the AS group and the mixture group were compared. The results showed that the total amount of amoxicillin, amoxicilloic acid, amoxicillin diketopiperazine, and the highest concentration of sulbactam in the liver and kidney samples of the AS group and the mixture group appeared at 1 h after drug withdrawal. Between 1 h and 12 h post discontinuation, the total amount of amoxicillin, amoxicilloic acid, and amoxicillin diketopiperazine in the two tissues decreased rapidly, and the elimination half-life of the AS group was significantly higher than that in the mixture group (p < 0.05); the residual amount of sulbactam also decreased rapidly, and the elimination half-life was not significantly different (p > 0.05). In 72 h urine samples, the total excretion rates were 60.61 ± 2.13% and 62.62 ± 1.73% in the AS group and mixture group, respectively. The total excretion rates of fecal samples (at 72 h) for the AS group and mixture group were 9.54 ± 0.26% and 10.60 ± 0.24%, respectively. These results showed that the total quantity of amoxicillin, amoxicilloic acid, and amoxicillin diketopiperazine was eliminated more slowly in the liver and kidney of the AS group than those of the mixture group and that the excretion rate through urine and feces was essentially the same for both groups. The residual elimination pattern of the hybrid molecule in rats determined in this study provides a theoretical basis for the in-depth development and application of hybrid molecules, as well as guidelines for the development of similar drugs.

Magnetic solid-phase extraction of amoxicillin and doxycycline from water and urine samples based on cetylpyridinium chloride-modified Fe3O4 nanoparticles followed by spectrophotometric determination.

This research describes an easy, rapid, and inexpensive magnetic solid-phase extraction (MSPE) approach employing Fe3O4 magnetic nanoparticles modified with cetylpyridinium chloride (Fe3O4@CPC/MNPs) for extracting amoxicillin (AMX) and doxycycline (DOX) after derivatization with 4-chloroaniline as a color reagent. The azo-coupling of AMX and DOX with the color reagent in the alkaline medium caused yellow and yellow-orange azo dyes with maximum absorption wavelengths of 435 and 438 nm, respectively. The UV-Vis spectroscopy was utilized to determine the target analyte after the extraction procedure. Good linearities (R2 > 0.99) in the concentration ranges of 0.03-4.50 and 0.05-6.00 µg/mL were obtained for AMX and DOX, respectively. The experimental detection limits of AMX and DOX were obtained as 0.01 and 0.02 µg/mL, respectively. The developed approach was effectively applied to pre-concentrate and quantify AMX and DOX in environmental water and urine samples.

Identification of Amoxicillin Crystals in Urine: a Case Report.

BACKGROUND: The case concerns a 30-year-old woman in the 24th week of pregnancy presenting to the medical emergency room with fever and abdominal pain. Urine sediment microscopy revealed the presence of unknown needle-shaped crystals. METHODS: Crystals identification was performed by Fourier-Transform Infrared Spectroscopy coupled to Attenuated Total Reflectance (FTIR-ATR). RESULTS: Amoxicillin crystals were verified with semiquantitative results of 87.7%. CONCLUSIONS: Drug-induced crystalluria is a frequent finding in urine examination and it may be asymptomatic. FTIR spectroscopy is a rapid and specific tool in identification of crystals and could be useful supporting renal disease diagnosis and monitoring drug therapy.

A new electrochemical platform based on low cost nanomaterials for sensitive detection of the amoxicillin antibiotic in different matrices.

A new electrochemical device based on a combination of nanomaterials such as Printex 6L Carbon and cadmium telluride quantum dots within a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate film was developed for sensitive determination of amoxicillin. The morphological, structural and electrochemical characteristics of the nanostructured material were evaluated using X-ray diffraction, confocal microscopy, transmission electron microscopy and voltammetric techniques. The synergy between these materials increased the electrochemical activity, the electron transfer rate and the electrode surface area, leading to a high magnitude of the anodic peak current for the determination of amoxicillin. The electrochemical determination of the antibiotic was carried out using square-wave voltammetry. Under the optimised experimental conditions, the proposed sensor showed high sensitivity, repeatability and stability to amoxicillin determination, with an analytical curve in the amoxicillin concentration range from 0.90 to 69 µmol L-1, and a low detection limit of 50 nmol L-1. No significant interference in the electrochemical signal of amoxicillin was observed from potential biological interferences and drugs widely used, such as uric acid, paracetamol, urea, ascorbic acid and caffeine. It was demonstrated that without any sample pre-treatment and using a simple measurement device, the sensor could be an alternative method for not only the analysis of pharmaceutical products (commercial tablets) and clinical samples (urine), but also to examine food quality (milk samples).

Antibiotic use and prescription and its effects on Enterobacteriaceae in the gut in children with mild respiratory infections in Ho Chi Minh City, Vietnam. A prospective observational outpatient study.

BACKGROUND AND OBJECTIVES: Treatment guidelines do not recommend antibiotic use for acute respiratory infections (ARI), except for streptococcal pharyngitis/tonsillitis and pneumonia. However, antibiotics are prescribed frequently for children with ARI, often in absence of evidence for bacterial infection. The objectives of this study were 1) to assess the appropriateness of antibiotic prescriptions for mild ARI in paediatric outpatients in relation to available guidelines and detected pathogens, 2) to assess antibiotic use on presentation using questionnaires and detection in urine 3) to assess the carriage rates and proportions of resistant intestinal Enterobacteriaceae before, during and after consultation. MATERIALS AND METHODS: Patients were prospectively enrolled in Children's Hospital 1, Ho Chi Minh City, Vietnam and diagnoses, prescribed therapy and outcome were recorded on first visit and on follow-up after 7 days. Respiratory bacterial and viral pathogens were detected using molecular assays. Antibiotic use before presentation was assessed using questionnaires and urine HPLC. The impact of antibiotic usage on intestinal Enterobacteriaceae was assessed with semi-quantitative culture on agar with and without antibiotics on presentation and after 7 and 28 days. RESULTS: A total of 563 patients were enrolled between February 2009 and February 2010. Antibiotics were prescribed for all except 2 of 563 patients. The majority were 2nd and 3rd generation oral cephalosporins and amoxicillin with or without clavulanic acid. Respiratory viruses were detected in respiratory specimens of 72.5% of patients. Antibiotic use was considered inappropriate in 90.1% and 67.5%, based on guidelines and detected pathogens, respectively. On presentation parents reported antibiotic use for 22% of patients, 41% of parents did not know and 37% denied antibiotic use. Among these three groups, six commonly used antibiotics were detected with HPLC in patients' urine in 49%, 40% and 14%, respectively. Temporary selection of 3rd generation cephalosporin resistant intestinal Enterobacteriaceae during antibiotic use was observed, with co-selection of resistance to aminoglycosides and fluoroquinolones. CONCLUSIONS: We report overuse and overprescription of antibiotics for uncomplicated ARI with selection of resistant intestinal Enterobacteriaceae, posing a risk for community transmission and persistence in a setting of a highly granular healthcare system and unrestricted access to antibiotics through private pharmacies. REGISTRATION: This study was registered at the International Standard Randomised Controlled Trials Number registry under number ISRCTN32862422: http://www.isrctn.com/ISRCTN32862422.

Development and validation of a method to determine amoxicillin in physiological fluids using micellar liquid chromatography.

A simple and robust method was developed for the routine identification and quantification of amoxicillin by micellar LC. Amoxicillin, a beta-lactamase inhibitor, is one of the most commonly prescribed drugs in the treatment of urine and skin structure infections. In this work, amoxicillin was determined in urine samples without any pretreatment step in a phenyl column using a micellar mobile phase of 0.10 M SDS and 4% butanol at pH 3. A UV detection set at 210 nm was used. Amoxicillin was eluted at 5.1 min with no interference by the protein band or endogenous compounds. Linearities (r >0.9998), intra- and interday precisions were determined (RSD (%) 0.4-2.7% and 0.3-5%, respectively, in micellar media, and 0.14-2.6% and 0.13-6%, respectively, in urine), and robustness was studied in the method validation. LOD and LOQ were 0.04 and 0.1 microg/mL in micellar media and 0.14 and 0.34 microg/mL in urine, respectively. Recoveries in the urine matrix were in the range of 95-110%. The validated method proved to be reliable and sensitive for the determination of amoxicillin in urine samples.

Selective solid-phase extraction of amoxicillin and cephalexin from urine samples using a molecularly imprinted polymer.

In this paper we describe, for the first time, a molecularly imprinted polymer (MIP) for the antibiotic amoxicillin (AMX), synthesised by a noncovalent molecular imprinting approach and used to extract AMX selectively from urine samples. The MIP was applied as a molecularly selective sorbent in molecularly imprinted SPE (MISPE) in an off-line mode, where it showed useful cross-selectivity for a structurally related antibiotic, cephalexin (CPX). By using a MISPE protocol, the MIP was able to selectively extract both AMX and CFX from 5 mL of water spiked with 10 mg/L with recoveries of 75 and 78% for AMX and CFX, respectively. When applied to real samples (urine) at clinically relevant concentrations, recoveries from 2 mL of human urine spiked with 20 mg/L decreased slightly to 65 and 63% for AMX and CFX, respectively. To demonstrate further the selectivity of the MIP obtained, a comparison with commercially available SPE cartridges was performed. Improvements in the retention of both AMX and CFX on the MIP were obtained relative to the commercially available cartridges, and the MISPE extracts were considerably cleaner, due to molecularly selective analyte binding by the MIP.

Pharmacokinetics of amoxicillin in human urine using online coupled capillary electrophoresis with electrogenerated chemiluminescence detection.

A novel and sensitive method for the determination of amoxicillin (AM) in human urine has been established using capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection, based on the ECL enhancement of Tris(2,2'-bipyridyl) ruthenium(II) with AM. The effects of several factors such as the detection potential, the concentration and the pH of phosphate buffer, the electrokinetic voltage and the injection time were investigated. Under the optimal conditions, the linear concentration of AM ranged from 1.0 ng/ml to 8.0 microg/ml (with a correlation coefficient of 0.9999). The limit of detection was 0.31 ng/ml. The mean recovery was 95.77% with relative standard deviations of no larger than 2.2%. This method is quick (the total run time within 6 min). This method has been successfully applied to a pharmacokinetic study in human urine after oral administration of AM.

Development of amoxicillin enzyme-linked immunosorbent assay and measurements of tissue amoxicillin concentrations in a pigeon microdialysis model.

A sensitive ELISA was developed for the detection of amoxicillin (AMX) in serum, urine, and milk. The ELISA used an indirect competitive method produced by coating the plate with ovalbumin conjugated with AMX hapten. Antibodies against AMX-BSA were detected by a goat-antirabbit antibody conjugated with peroxidase. Calibration standard curves ranged from 1.28 ng/mL to 20 microg/mL [IC(50) (inhibition concentration 50%) = 100 ng/mL], and the limits of detection were 1.3, 2.7, and 4.8 ng/mL for urine, milk, and serum, respectively. The intra- and interassay variations were less than 4 and 9.6%. The antibody produced against AMX cross-reacted highly with penicillin G (77%); cross-reacted moderately with ampicillin, oxacillin, and cloxacillin (56.9, 51.4, and 48.8%, respectively); but was considered non-cross-reactive with dicloxacillin (7.4%), cefadroxil (<1%), and cefazolin (<1%). Concentrations of AMX were measured simultaneously in venous blood and muscles by using the developed AMX ELISA in an in vivo microdialysis model designed for pigeons. Following i.m. injection (25 mg/kg), AMX attained a peak blood level of 4.74 +/-0.30 mu g/mL and decreased with a half-life of 2.38 +/-0.16 h. In contrast, measurements in pectoral and femoral muscles exhibited delayed appearances, reduced peak concentrations, and prolonged half-lives of 4.07 +/-0.48 (pectoral) and 3.01 +/-0.26 (femoral) that were significantly different from each other and those in the blood (P < 0.05). Blood protein binding was calculated to be 27.9 +/-5.7%. This study demonstrated the semiquantitative application of a selective AMX ELISA in the first microdialysis procedure for continuous monitoring of drug levels in specific tissues of pigeons and maybe useful for related studies in other poultry species.

Use of Polymer Inclusion Membranes (PIMs) as support for electromembrane extraction of non-steroidal anti-inflammatory drugs and highly polar acidic drugs.

The use of polymer inclusion membranes (PIMs) as support of 1-octanol liquid membrane in electromembrane extraction (EME) procedure is proposed. Synthesis of PIMs were optimized to a composition of 29% (w/w) of cellulose triacetate as base polymer and 71% (w/w) of Aliquat®336 as cationic carrier. Flat PIMs of 25µm thickness and 6mm diameter were used. EME protocol was implemented for the simultaneous extraction of four non-steroidal anti-inflammatory drugs (NSAIDs) (salicylic acid, ketoprofen, naproxen and ibuprofen) and four highly polar acidic drugs (anthranilic acid, nicotinic acid, amoxicillin and hippuric acid). Posterior HPLC separation of the extracted analytes was developed with diode array detection. Recoveries in the 81-34% range were obtained. EME procedure was applied to human urine samples.

Hydrophilic interaction chromatography coupled to nuclear magnetic resonance spectroscopy and mass spectroscopy--a new approach for the separation and identification of extremely polar analytes in bodyfluids.

A method for the unambiguous identification of highly polar molecules based on the separation on a silica gel column run in hydrophilic interaction chromatography (HILIC) mode followed by mass spectroscopic (MS) analysis and subsequent measurement by nuclear magnetic resonance (NMR) spectroscopy is described. Polar neutral, acidic and basic compounds of small molecular size usually not retained on reversed phase stationary phases can be separated and unequivocally identified by means of MS and NMR spectroscopy. The method is applied to exemplify the identification of the endogenous metabolite trigonelline and the polar antibiotic amoxicilline in human urine.

Surfactant coated fullerenes C60 as pseudostationary phase in electrokinetic chromatography.

In the present paper, surfactant coated fullerenes C(60) (SC-C(60)) have been proposed as a novedous pseudostationary phase to improve separation of different aromatic compounds. The target analytes were beta-lactams antibiotics, non-steroidal anti-inflammatory drugs and amphenicols. In all cases, the analytes interacted with the pseudostationary phase producing an important enhancement on resolution. The results were compared with those obtained with surfactant coated carbon nanotubes (single-walled and multi-walled nanotubes), showing that in the proposed conditions, fullerenes C(60) were advantageous as interactions between the analytes and the pseudostationary phase were more effective. Finally, the procedure was applied to pharmaceuticals and urine samples, with satisfactory results.

Comprehensive study on the electrocatalytic effect of copper - doped nano-clinoptilolite towards amoxicillin at the modified carbon paste electrode - solution interface.

In this work, Cu(II)-exchanged clinoptilolite nanoparticles (Cu(II)-NCL) were prepared and characterized by FTIR, BET, XRD and TEM techniques. The obtained Cu(II)-NCL was then used for the modification of carbon paste electrode (CPE). The resulted Cu(II)-NCL/CPE was finally used for the voltammetric determination of amoxicillin (AMX). The best voltammetric response was obtained by the electrode containing 20% of the modifier in 0.05molL-1 NaCl at pHs 2.2 and 7.2. The electrode showed a linear response in the concentration range of 4.0×10-8-1.0×10-4molL-1 AMX with a detection limit of 2.0×10-8molL-1 in square wave voltammetry. The electrode showed good repeatability, reproducibility, long life time confirmed by statistical tests. The electrode had also good selectivity together with good applicability in determination of AMX in urine and pharmaceutical tablets as real samples.

New nanostructured support for carrier-mediated electromembrane extraction of high polar compounds.

A new support has been proposed to be used for carrier-mediated electromembrane extraction purposes. The new support (Tiss®-OH) is a 100µm thickness sheet nanofiber membrane manufactured by electrospinning and composed by acrylic nanofibers. It has been used in an electromembrane extraction (EME) combined with a HPLC procedure using diode array detection. The proposed method has been used for the extraction of four high polarity acidic compounds: nicotinic acid, amoxicillin, hippuric acid and salicylic acid. Analytes were extracted from an aqueous sample solution (pH 4) (donor phase) using a Tiss®-OH sheet that supports a 5% (w/v) Aliquat®336 in 1-octanol liquid membrane. Aqueous solution (pH 6) was used as acceptor phase. The electrical field was generated from a d.c. electrical current of 100V through two spiral shaped platinum wires placed into donor and acceptor phases. Analytes were extracted in 10min with recoveries in the 60-85% range. The proposed EME procedure has been successfully applied to the determination of the target analytes in human urine samples.

Acute renal failure secondary to drug-related crystalluria and/or drug reaction with eosinophilia and systemic symptom syndrome in a patient with metastatic lung cancer.

Drug reaction with eosinophilia and systemic symptoms (DRESS) or drug-induced hypersensitivity is a severe adverse drug-induced reaction. Aromatic anticonvulsants, such as phenytoin, phenobarbital, and carbamazepine, and some drugs, can induce DRESS. Atypical crystalluria can be seen in patients treated with amoxycillin or some drugs and can cause acute renal failure. We describe a 66-year-old man who presented fever and rash and acute renal failure three days after starting amoxycillin. He was also using phenytoin because of cerebral metastatic lung cancer. Investigation revealed eosinophilia and atypical crystalluria. The diagnosis of DRESS syndrome was made, amoxicillin was stopped, and dose of phenytoin was reduced. No systemic corticosteroid therapy was prescribed. Symptoms began to resolve within three to four days. The aim of this paper is to highlight the importance of microscopic examination of urine in a case with acute renal failure and skin lesions to suspect DRESS syndrome.

Solid phase extraction of amoxicillin using dibenzo-18-crown-6 modified magnetic-multiwalled carbon nanotubes prior to its spectrophotometric determination.

This work reports on a method for selective extraction and sensitive determination of amoxicillin drug (AMX). The method is based on solid phase extraction of AMX by a novel modified magnetic nanoadsorbent prior to spectrophotometric determination of AMX using a procedure based on formation a colored azo-derivative of the investigated drug. The nanoadsorbent has been synthesized by modification of magnetic-multiwalled carbon nanotube with dibenzo-18-crown-6 moieties. The synthesized nanoparticles were characterized using TEM, XRD and FT-IR measurements. At the next step, various factors that could potentially affect adsorption and desorption efficiencies of AMX, have been optimized. The results showed that under the optimized conditions, sensitive and selective determination of the investigated drug in concentration range of 5.0-1000.0 ng mL(-1) with the limit of detection of 3.0 ng mL(-1) was achievable. Furthermore, the real sample analysis (i.e. amoxicillin capsules and human urine samples) results indicated that a reliable promising candidate method has been developed for the determination of AMX in the investigated real samples.