A selective and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the measurement of colistin residues in milk and in four bovine tissues (i.e., muscle, liver, kidney, and fat). The sample treatment consists of protein precipitation using 10% (w/v) trichloroacetic acid, solid-phase purification on C18 cartridges, and precolumn derivatization of colistin with ortho-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). This latter step is performed automatically, and the resulting reaction mixture is injected into a switching HPLC system including a precolumn and an analytical column packed with end-capped LiChrospher RP18 (5 microns). Washing the precolumn and final elution onto the analytical column are conducted using acetonitrile-0.01M phosphate buffer (pH 7.0) mixtures with respective proportions of 65:35 and 68:32 (v/v). Detection is carried out by spectrofluorometry (excitation wavelength, 340 nm; emission wavelength, 440 nm). The retention times of the derivatives corresponding to the two main components of colistin (i.e., polymyxins E2 and E1) are approximately 14 and 18 min, respectively. The structural study of the derivatives corresponding to polymyxins E1 and E2 is carried out by HPLC coupled with electrospray mass spectrometry; data obtained confirms that the derivatization process occurs with the five amino groups of the analytes. Selectivity is obtained in the HPLC system versus other coadministered anti-infective drugs (beta-lactams, aminoglycosides, tetracyclines, and sulphonamides) and endogenous compounds. Quantitation is performed using the sum of the peak areas of polymyxin E1 and polymyxin E2 derivatives. Testing linearity affords correlation coefficients greater than 0.990 for calibration curves in the range of 10-500 microL/L for milk, 50-1000 micrograms/kg for muscle and fat, and 100-1000 micrograms/kg for kidney and liver. Relative standard deviation values are less than 10% at a concentration of 25 micrograms/L in milk and 100 micrograms/kg in tissues (six replicates); recoveries are higher than 60%.

Hyphenated Liquid Chromatographic Method for the Determination of Colistin Residues in Bovine Tissues

Sample preparation for residue determination of gentamicin and neomycin by liquid chromatography.

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used in veterinary medicine for the treatment of serious infections. The purpose of this study was to develop and validate a method to determine gentamicin residues in edible tissues of swine and calf. Extraction of gentamicin was performed using a liquid extraction with phosphate buffer containing trichloroacetic acid, followed by a solid-phase clean-up procedure on a CBA weak cation-exchange column. Tobramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS). Chromatographic separation of the internal standard tobramycin and the gentamicin components was achieved on a Nucleosil (5 µm) column using a mixture of 10 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. The gentamicin components C1a, C2 + C2a and C1 could be identified with the MS/MS detection, and subsequently quantified. The method was validated according to the requirements of the EC at the maximum residue limit (MRL) (100 ng g−1 for muscle and fat, 200 ng g−1 for liver and 1000 ng g−1 for kidney), half the MRL and double the MRL levels. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit <10%). Limits of quantification of 25.0 ng g−1 were obtained for the determination of gentamicin in muscle, fat, liver and kidney tissues of swine and calf, which correspond in all cases to at least half the MRLs. Limits of detection ranged between 0.5 and 2.5 ng g−1 for the tissues. The within-day and between-day precisions (RSD) and the results for accuracy fell within the ranges specified. The method was successfully used for the determination of gentamicin in tissue samples of swines and calves medicated with gentamicin by intramuscular injection. Copyright © 2000 John Wiley & Sons, Ltd.

Determination of gentamicin in swine and calf tissues by high-performance liquid chromatography combined with electrospray ionization mass spectrometry.

Four simple, accurate, sensitive and economical procedures (A–D) for the estimation of gentamicin sulphate and vancomycin hydrochloride, both in pure form and in pharmaceutical formulations have been developed. The methods are based on the oxidation of the studied drugs by a known excess of potassium permanganate in sulphuric acid medium and subsequent determination of unreacted oxidant by reacting it with amaranth dye (method A), acid orange II (method B), indigocarmine (method C) and methylene blue (method D), in the same acid medium at a suitable λmax=521, 485, 610 and 664 nm, respectively. The reacted oxidant corresponds to the drug content. Regression analysis of Beer-Lambert plots showed good correlations in the concentration ranges 4–8, 3–8, 4–9 and 5–9 µg ml−1 with gentamicin and 4–8, 1.5–4, 1.5–4 and 3.5–5.5 µg ml−1 with vancomycin for methods A, B, C, and D, respectively. The molar absorptivity, sandell sensitivity, detection and quantification limits were calculated. The stoichiometric ratios for the cited drugs were studied. The optimum reaction conditions and other analytical parameters were evaluated. The influence of the substance commonly employed as excipients with these drugs were studied. The proposed methods were applied to the determination of these drugs in pharmaceutical formulations. The results have demonstrated that the methods are equally accurate and reproducible as the official methods.

https://www.degruyter.com/downloadpdf/j/chem.2006.4.issue-4/s11532-006-0035-z/s11532-006-0035-z.xml

Indirect spectrophotometric determination of gentamicin and vancomycin antibiotics based on their oxidation by potassium permanganate

A simple and sensitive HPLC method was developed for the determination of colistin antibiotic in feeds employing pre-column derivatization and fluorescence detection. Extraction of colistin in feeds was by sonication and shaking with 0.1 M HCl. Pre-column derivatization was with phthaldialdehyde and 2-mercaptoethanol (ME) in borate buffer (pH 10.5) to obtain a fluorescent derivative. Elution of the derivative onto an Ultracarb 5 μm ODS column was by using acetonitrile—ultrapure water (75∶25). Detection was by spectrofluorimetry at 340 nm (excitation wavelength) and 440 nm (emission wavelength). Total elution time was <20 min. The applicability of the validated method was tested by analyzing commercial medicated feeds without any interference from the matrix.

Simple HPLC determination of colistin in medicated feeds by pre-column derivatization and fluorescence detection

Development and optimization of a liquid chromatographic method for the determination of gentamicin in calf tissues

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Development and optimization of a liquid chromatographic method for the determination of gentamicin in calf tissues