Lincomycin analysis

For efficient extraction of macrolide and lincosamide residues from edible animal products, bound residues should be rendered soluble, most if not all of the proteins should be removed, and high recoveries for all analytes should be provided. Since tliese antibiotics do not strongly bind to proteins, many effective extraction methods have been reported. Sample extraction/deproteinization is usually accomplished by vortexing liquid samples or homogenizing semisolid samples with acetonitrile (136—139), acidified (136,140-142) orbasified acetonitrile (143), methanol (14, 144, 145), acidified (145-147) or basified methanol (148), chloroform (149-151), or dichloromethane under alkaline conditions (152). However, for extraction of sedecamycin, a neutral macrolide antibiotic, from swine tissues, use of ethyl acetate at acidic conditions has been suggested (153), while for lincomycin analysis in fish tissues, acidic buffer extraction followed by sodium tungstate deproteinization has been proposed (154). 

A number of small molecules of interest in clinical analyses have also been analyzed using microchip-based separations. Zhao et al. utilized a microchip separation for detection on the antibiotic lincomycin directly from urine samples. This method, also employing electrochemical detection, allowed lincomycin analysis in under 40 s. Crevillen et al. utilized a similar method for... 

Krzek et al. [35] reported the qualitative identification and quantitative analysis of the mixtures of OTC, tiamulin, lincomycin, and spectinomycin in the veterinary preparations by using TLC/densitometry. As stationary phase, they used precoated TLC aluminum sheets, and the mobile phases were mixtures of 10% citric acid solution, hexane, ethanol (80 1 1, v/v), and n-butanol, ethanol, chloroform, 25% ammonia (4 5 2 5, v/v). The other application of TLC or HPTLC for analyzing OTC in the various samples is summarized in Table 2 [36]. 

Separations were performed on either silica- or amino-bonded stationary phase columns, using carbon dioxide with various modifiers as mobile phases. Each column exhibited distinctly different selectivities to the examined sulfonamides, the amino-bonded column being much more sensitive to modifier variations. In a continuation of the sulfonamide study, packed-column SFC was further evaluated for possible application to the analysis of furazolidone, chloramphenicol, and lincomycin residues (82). Separation was effected on an amino-bonded stationary phase using carbon dioxide with methanol modifier as the mobile phase, whereas detection was accomplished by MS. 

In traditional electrophoresis, separation efficiency is limited by thermal diffusion and convection. Owing to long analysis times and low efficiencies, these procedures never enjoyed wide usage. Problems have arisen when trying to differentiate between structurally related drug residues such as streptomycin and dihydrostreptomycin, tetracyclines, lincomycin and clindamycin, and erythromycin and oleandomycin (83, 84). To overcome these problems, anticonvective media, such as polyacrylamide or agarose gels, have also been used. 

In liquid chromatographic analysis of macrolides and lincosamides, most popular is the ultraviolet detector (Table 29.4). Tylosin, tilmicosin, spiramycin, sedecamycin, and josamycin exhibit relatively strong ultraviolet absorption, but erythromycin, lincomycin, pirlimycin, and oleandomycin show extremely weak absorption in the ultraviolet region. Hence, detection at 200-210 nm has been reported for the determination of lincomycin (146). However, a combination of poor sensitivity and interference from coextractives necessitated extensive cleanup and concentration of the extract. Precolumn derivatization of pirlimycin with 9-fluorenylmethyl chloroformate has also been described to impart a chromophore for ultraviolet detection at 264 nm (140). 

Electrochemical detection is better suited to the analysis of erythromycin and lincomycin. This method of detection has been applied for the determination of erythromycin A (139) and lincomycin (154) residues in salmon tissues. Liquid chromatography coupled with mass spectrometry is particularly suitable for confirmatory analysis of the nonvolatile macrolides and lincosamides. Typical applications of this technique are through thermospray mass spectrometry, which has been used to monitor pirlimycin in bovine milk and liver (141,142), and chemical ionization, which has been applied for identification of tilmicosin (151) in bovine muscle, and for identification of spiramycin, tylosin, tilmicosin, erythromycin, and josamycin residues in the same tissue (150). 

Neville, G. A. J. C. Ethier, N. F. H. Bright, and R. H. Lake. 1971. Characterization of some lincomycin and cyclamate salts by thermal analysis and infrared spectrosAc soc. Off. Anal. Cheifi4 1200-1210. 

C NMR Spectrum and Spin - Lattice relaxation times were used to study the structure of lincomycin (24). The results of analysis are reported in Table (III) and Figure (V). The signal assignments were based on spectral comparison of the structured related compounds and on other known techniques. 

Near infra red reflectance analysis via Mahalanobis distance determination was found to be a useful technique for determining the concentration of lincomycin in an agricultural premix. The results obtained were comparable to the corresponding values collected with the use of the reference sample. 

TLC Silica gel plate carrying lincomycin has been developed in 80/20 chloroform / methanol mixture and sprayed with 5% Vanillin In methanol and 20% w/v sulfuric acid in methanol (28). The plate was gently heated to observe lincomycin yellow spots. The Rf of lincomycin is 0.5. To quantitate samples, the plates were heated until red brown spots developed which were stable and amenable to TLC direct analysis. Tenths of micrograms of lincomycin were detectable by this method (28). 

Lincomycin in plasma (human or povine) and urine determined by HPLC at 214 nm using ion - pair HPLC column with an acetonitrile - trifluoroacetic acid ( 0.1 - 0.2 % v/v in the mixture) as an eluent (33). More than 1800 samples were assayed by this method. Soil - phase extraction (SPE) procedure was used in this analysis followed by an evaporation step. 

A microbiological assay was described for lincomycin in animal feed using an agar diffusion assay and an agar slant of Sarcina Lutea ATCC No. 9341 as the test organism for the analysis of lincomycin (42). 

Lincomycin hydrochloride and aminoglycoside hydrochloride were determined in pharmaceuticals by Isotachophoresis. In this method, a mixture of 0.02 M potassium acetate and 0.3 % hydroxypropylmethyl cellulose (15,000) was used as an aqueous electrolyte system, whereas a mixture of 20um 1,4-aminobutyric acid in acetic acid (pH 4.72) or 0.02 M glycylglycine or B -alanine was used as the terminator. The analysis was performed at a constant current of 200 jjA at 5°C. it was found that the minimum amount that could be quantitated by this method was 1.6 nmol and the relative standard deviation was 2%. 

Lincomycin (and to some extent the other lincosamides) is commonly mislabeled as a macrolide and as such is often included in multi-residue macrolide procedures. Honey has been the most common target matrix, because of interest in the use of lincomycin for control of foulbrood disease in bees. Extraction from honey has principally been by dissolution/dilution with aqueous solvents. Acetonitrile has been used as extractant for tissues and milk. Dispersion onto sand prior to hot-water extraction has also been used for the analysis of milk. Solid-phase " and liquid-liquid extraction have been used for clean-up. Separation is customarily achieved on reversed phase (C18) columns prior to determination by MS or MS/MS with APCI or more commonly ESI. ... 

Sin DWM, Wong YC, Ip ACB, Quantitative analysis of lincomycin in animal tissues and bovine milk by liquid chromatography electrospray ionisation tandem mass spectrometry, J. Pharm. Biomed. Anal. 2004 34(3) 651-659. 

Discriminant analysis of NIR spectra was used in 1986 to assay the level of lincomycin in a pharmaceutical formulation. This was the first NIR analysis to be accepted by this US Food and Drug Administration (FDA). NIR is now used to measure the salicylic acid content of aspirin and pharmaceutical companies use discriminant NIR procedures to check incoming raw materials for drug production. Sample identification can be achieved using at-line NIR fiber optic systems. 

In the following year, lincomycin was determined by the microchip CE-ECL system where ITO working electrode was fabricated by photolithographic method from an ITO-coated glass slide (chip substrate) located at the end of the separation channel (Fig. 5.10). The top layer made up of a poly(dimethylsiloxane) (PDMS) layer consisting of two channels, namely separation and injection channels. This microchip CE-ECL system can be successfully applied for the rapid analysis of... 

Several antibiotics of clinical and pharmaceutical importance were also determined on the basis of ECL detection method. Eor instance, based on the enhancing effect of clarithromycin on ECL of Ru(bpy)3 after CE separation, a new analytical method for sensitive determination of clarithromycin in biological fluids was developed and a detection limit of 30 nM was achieved. The proposed method has been successfully applied for clarithromycin content determination in spiked human plasma and urine samples [50]. Lincomycin was determined in the urine sample by microchip CE with integrated ITO working electrode based on ECL detection. This microchip CE-ECL system can be used for the rapid analysis of lincomycin within 40 s. This method is promising for detection of lincomycin in clinical and pharmaceutical area [51]. A novel method for ECL detection from... 

Preliminary data have been obtained for the putative transthiomethylation of Cl (Rolls JP and Messier EJ, unpublished data). The S. lincolnemis culture was fed S] DL-methionine under conditions of lincomycin production. The analysis of the lin-comycin showed equal incorporation of from the [ CHj. S]-DL-methionine into... 

Quantitative analysis of tablets and other dosage forms were also performed early in the current wave of NIR. In 1982, Rose et al. reported the direct analysis of meglumine and meglumine diatrizoate in injectable solutions, for example." In 1986, Whitfield developed a method to measnre the amount of lincomycin in granulations for veterinary purposes. This was the first NIR method accepted by the FDA as a primary method." ... 

The FDA has recognized the value of NIR spectroscopy, and has already approved the method for the analysis of lincomycin in a veterinary product [136]. More recently, NIR spectroscopy has been employed in the qualification of incoming raw materials, and the FDA is working with pharmaceutical manufacturers to develop and implement other NIR-based methods, with several having been approved. 

An analysis of the many cases reported in the literature has shown that there appear to be two groups of patients showing these side effects. Group 1 comprises patients who develop diarrhoea or mild colitis at an early stage of treatment with the lincomycins. These reactions usually appear soon after treatment has started and disappear after the antibiotic has been withdrawn. This self-limit-... 

Orthopaedic hospital patients often need lincomycin or clindamycin therapy an analysis of 1158 of these patients treated with antibiotics revealed 58 cases of diarrhoea, many of them in association with lincomycin, as well as several cases during treatment with other antibiotics such as the penicillins and tetracyclines. Since in this study there were only 3 documented cases of lincomycin-associated colitis, the conclusion was drawn that continued use of lincomycin or clindamycin appears to be fully justified by the low incidence of colitis (49 -, 50 ). In another study there have been 3 severe cases out of 144 patients receiving lincomycins as a prophylactic measure for total hip replacements. It is concluded in this study that as these drugs may produce a lethal condition, they should be used with appropriate discretion, especially in the elderly female who appears to be most at risk (48 ). 

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