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.

Combination of a sub-3 µm superficially porous particle packed column with charged aerosol detection for the simple and sensitive measurement of nine macrolides in human urine.

Due to the lack of chromophores in many macrolides, analytical methods based on mass spectrometry and electrochemical detection coupled to liquid chromatography have been suggested to be suitable for the quantification of macrolides in complex matrices. In this study, a simple and sensitive analytical method was established for the simultaneous measurement of nine macrolides in human urine by combining a sub-3 µm superficially porous particle packed column with charged aerosol detection. After thorough investigation of various sample preparation methods, including two liquid-liquid extraction methods and four solid-phase extraction methods, HLB solid-phase extraction was selected and further optimized. Absolute recovery of the optimized sample preparation method ranged from 99.5-110.2%, indicating its very high extraction/clean-up efficiency. For chromatography, parameters influencing macrolide separation were systematically optimized, and the resulting conditions allowed baseline separation of nine macrolides within 24 min using a very simple mobile phase. The established method was validated for linearity, limit of detection, limit of quantification, absolute recovery, and precision. Based on its limit of detection (0.025-0.100 µg/mL), the method had similar or greater sensitivity than most methods based on electrochemical detection. It was found that the current method was appropriate for application to real human urine samples after drug administration.

Combined application of dispersive liquid-liquid microextraction based on the solidification of floating organic droplets and charged aerosol detection for the simple and sensitive quantification of macrolide antibiotics in human urine.

A novel analytical method combining dispersive liquid-liquid microextraction based on the solidification of floating organic droplets (DLLME-SFO) and liquid chromatography with charged aerosol detection (LC-CAD) was established. For the first time, CAD was applied for the detection of macrolide antibiotics lacking chromophores. Parameters influencing the microextraction efficiency were systematically investigated, and the optimized microextraction conditions yielded high enrichment factors in the range of 60-106. The combined application of DLLME-SFO and LC-CAD provided the sensitivity of the method, expressed as the limit of detection (LOD), as low as 10 to 40ngmL(-1) and intra-day and inter-day precisions below 8.7% and 12.6%, respectively. The measured absolute recovery values were approximately 100%, indicating that the extraction efficiency was very high. Direct comparisons of the liquid-liquid extraction and organic solvent precipitation methods demonstrated that the proposed method was more sensitive, specific, rapid, and environmentally friendly for the determination of five macrolide antibiotics in human urine. The results suggest that the combined use of DLLME-SFO and LC-CAD may be applicable to the analysis of various compounds with poor to no chromophores in complex matrices.

A simple and sensitive HPLC-UV determination of 7-O-succinyl macrolactin A in rat plasma and urine and its application to a pharmacokinetic study.

A simple, sensitive and reproducible isocratic reversed-phase (C(18) ) high-performance liquid chromatography (HPLC) method was developed to determine 7-O-succinyl macrolactin A (SMA) in rat plasma and urine samples using UV detector set at 230 nm. Lamotrigine was used as internal standards (IS) to ensure the precision and accuracy of the method. The retention times of SMA and IS for the plasma sample were 9.2 and 4.4 min, respectively, and those for the urine samples were 7.9 and 4.3 min, respectively. The intra- and inter-day variations of the analytical responses, expressed in terms of relative standard deviation, were less than 14.9%. The accuracy, in terms of average analytical recovery, ranged from 90.4 to 119%. The lower limits of quantification of SMA in rat plasma and urine samples were 0.02 and 0.1 µg/mL, respectively. This method is applicable for the pharmacokinetic studies of SMA in rats.

Determination of macrolide antibiotics using dispersive liquid-liquid microextraction followed by surface-assisted laser desorption/ionization mass spectrometry.

A novel method for the determination of macrolide antibiotics using dispersive liquid-liquid microextraction coupled to surface-assisted laser desorption/ionization mass spectrometric detection was developed. Acetone and dichloromethane were used as the disperser solvent and extraction solvent, respectively. A mixture of extraction solvent and disperser solvent were rapidly injected into a 1.0 mL aqueous sample to form a cloudy solution. After the extraction, macrolide antibiotics were detected using surface-assisted laser desorption/ionization mass spectrometry (SALDI/MS) with colloidal silver as the matrix. Under optimum conditions, the limits of detection (LODs) at a signal-to-noise ratio of 3 were 2, 3, 3, and 2 nM for erythromycin (ERY), spiramycin (SPI), tilmicosin (TILM), and tylosin (TYL), respectively. This developed method was successfully applied to the determination of macrolide antibiotics in human urine samples.

A sensitive method for simultaneous determination of four macrolides by CE with electrochemiluminescence detection and its applications in human urine and tablets.

A sensitive method for simultaneous determination of azithromycin (AZI), acetylspiramycin (ACE), erythromycin (ERY), and josamycin (JOS) was developed by CE coupled with electrochemiluminescence detection with Ru(bpy)(3) (2+). The parameters related to separation and detection were investigated in detail. The four macrolides were well separated and detected within 6 min under the optimized conditions. The LOD (S/N=3) of AZI, ACE, ERY, and JOS were 1.2 x 10(-9), 7.1 x 10(-9), 3.9 x 10(-8) and 9.5 x 10(-8) mol/L, respectively. The LOQ (S/N=10) of AZI, ACE, ERY, and JOS in human urine were 8.2 x 10(-8), 2.5 x 10(-7), 8.9 x 10(-7) and 1.2 x 10(-6) mol/L, respectively. The recoveries of the four macrolides in human urine and pharmaceutical tablet samples were 85.0-104.0% at different concentration levels.

Spectrophotometric determination of four macrolide antibiotics in pharmaceutical formulations and biological fluids via binary complex formation with eosin [corrected].

A simple, rapid, and sensitive validated spectrophotometric method was developed for the determination of certain macrolide antibiotics namely, erythromycin (I), azithromycin dihydrate (II), clarithromycin (III), and roxithromycin (IV) in bulk powders, pharmaceutical formulations, and spiked biological fluids. The proposed method is based on the formation of a binary complex between each of the studied drugs and eosin Y in aqueous buffered medium. Under the optimum conditions, the binary complexes showed absorption maxima at 542-544 nm. The absorbance of the binary complexes obeyed Beer's law over the concentration range of 1-10 micro/g/mL for II, 2-20 microg/mL for I and IV, and 3-30 microg/mL for III. The mean percentage recoveries were 100.04 +/- 0.83, 99.98 +/- 0.80, 100.17 +/- 0.91, and 99.55 +/- 0.91, with minimum detectable molarities of 2 x 10(-7) for I and II, 4 x 10(-7) for III, and 3 x 10(-7) for IV. The different experimental parameters affecting the development and stability of the colors were studied and optimized. The proposed method was successfully applied to the analysis of the cited drugs in some pharmaceutical formulations. The results obtained were in good agreement with those obtained using the reference methods. The proposed method was further applied to spiked human urine and plasma. A proposal of the reaction pathway is suggested.

A validated spectrofluorometric assay for the determination of certain macrolide antibiotics in pharmaceutical formulations and spiked biological fluids.

This paper describes a simple spectrofluorometric method for the analysis of 4 macrolide antibiotics. The method is based on the condensation of 10% (w/v) malonic acid and acetic acid anhydride under the catalytic effect of tertiary amine groups of the studied macrolides. The relative fluorescence intensity of the condensation product was measured at 397/452 nm (excitation/emission) for azithromycin dihydrate and at 392/445 nm (for clarithromycin, erythromycin ethylsuccinate, and roxithromycin. All variables affecting the reaction conditions were studied. The effects of potential interference due to common excipients, such as starch, lactose, sucrose, glucose, gum acacia, and magnesium stearate, as well as trimethoprim and sulfisoxazole acetyl formulated in primomycin capsules and pediazole oral suspension, respectively, were studied. A validation study for the proposed method was carried out according to U.S. Pharmacopeia 2002. The linearity ranges were 3-80 ng/mL for all of the cited macrolides. The limit of detection range was 0.74-1.20 ng/mL, while the limit of quantitation range was 2.47-4.02 ng/mL. The method was applied for the assay of the studied macrolides in pure pharmaceutical formulations and in spiked biological fluids. Results were compared with those obtained from the reported method, where calculated t- and F-values indicated high accuracy and good precision for the proposed method.

Voltammetric investigation of macrolides by an HPLC-coulometric assay.

Voltammograms of macrolides, including anhydroerythromycin A, azithromycin, erythromycin A, erythromycin A enol ether, pseudoerythromycin A enol ether, oleandomycin and tylosin have been investigated using a dual electrode cell in combination with a high-throughput LC method. The half-wave potentials (E(1/2)) of the seven macrolides investigated ranged from 0.734 to 0.866 V, and the current responses reached the maxima at over 1.0 V. The current response of the downstream electrode displayed a non-linear behavior at high potentials over +0.75 V, probably because of polarization of solvent components, e.g., water. The HPLC-coulometric assay was optimized with the potentials of the upstream and downstream electrodes at +0.65 and +0.85 V, respectively. This method is suitable for detection of 14- and 15-membered macrolides (sensitivity<0.05 microg ml(-1)), but not for a 16-membered macrolide, tylosin (sensitivity>0.1 microg ml(-1)). The assay shows interferences from biomatrices in rat's blood plasma and serum, and human urine, but they were effectively removed by a cold acetonitrile extraction method.

Comparative study of coulometric and amperometric detection for the determination of macrolides in human urine using high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) method using chromatographic conditions optimised in a previous work was applied for the separation of three macrolide antibiotics roxithromycin (Rox), oleandomycin (Ole) and rosamicin (Ros) and further determination of two of them, roxithromycin (Rox) and oleandomycin (Ole), in human urine samples. A comparative study of the behaviour of these macrolides under the two types of electrochemical detection (EC) widely coupled with HPLC, that is coulometric (EC-C) and amperometric (EC-A), was carried out by applying the same multiresidue method. From the assays performed using both detectors the comparison was made taking relevant criteria such as detection limits, linearity, recovery and precision values into account. As a result of this comparison, the coulometric detector appears slightly more suitable than the amperometric one for macrolide analysis.