Analysis of Antibiotics

Immunoassays are commercially available for frequently monitored antibiotics including amikacin, gentamicin, tobramycin, and vancomycin. However, GC and in most cases HPLC assays are also available for determination of concentrations of antibiotics in biological matrix. Owing to lack of volatility, chromophore, and hydrophilicity of aminoglycosides, most methods require derivatization of these drugs before analysis. Packed column separation was generally used for GC methods. However, for HPLC methods, reverse phase, ion pair, ion exchange, and normal phase separation protocols have been reported (43). Lai and Sheehan (44) described a reversed-phase HPLC method for determination of tobramycin using pre-column derivatization with o-phthalaldehyde followed by fluorescence detection. Another HPLC method for determination of amikacin used pre-column derivatization of amikacin with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatization reaction can be conducted in aqueous medium at room temperature with borate buffer at pH 8.0. The formation of the derivative is instantaneous, and the derivative is stable for more than 36 h. Detection was performed by using UV absorption instead of fluorescence (45). Nicoli and Santi used 1-fluoro-2,4-dinitrobenzene for derivatizing amikacin for analysis by HPLC using a C18 column and mobile phase composition of acetonitrile-water-acetic acid (47:53:0.1 by volume). The column temperature was maintained at 45°C (46). Kawamoto et al. described an HPLC protocol for determination of serum concentrations of sisomicin, netilmicin, astromicin, and micronomicin using an amino acid analyzer system. The aminoglycosides were separated by reverse phase ion-pair chromatography on Zorbax C8 and ODS columns and detected by post column derivatization technique (47). Soltes (48) reviewed HPLC techniques for determination of aminoglycoside concentrations in biological specimens developed in last two decades.

Aminoglycosides can also be determined by HPLC technique without derivatization using evaporative light scattering detection technique (49). Some investigations also used liquid chromatography combined with mass spectrometry for analysis of aminoglycosides. Kim et al. analyzed kanamycin, neomycin, and gentamicin after deriva-tization with phenylisocyanate. The authors used a liquid chromatography combined with electrospray ionization mass spectroscopy for separation and final analysis of these aminoglycosides (50). An analytical method using liquid chromatography combined with mass spectrometry has also been reported for simultaneous determination of metronidazole and spiramycin I in human plasma, saliva, and gingival crevicular fluid. Ornidazole was used as the internal standard. After liquid-liquid extraction, drugs were analyzed using a 5-^m Kromasil C18 column (150 x 4.6 mm ID, particle size 5 ^m) and a mobile phase composed of acetonitrile, water, and formic acid with a solvent gradient (51). Amphotericin B is used for the treatment of invasive and disseminated fungal infections. Lee et al. described a sensitive liquid chromatography tandem mass spectrometric method for quantification of total and free amphotericin B in biological matrices. The authors used a C18 column for HPLC analysis (52). Azithromycin in serum can be analyzed by HPLC using electrochemical detection. Fouda and Schneider described an HPLC combined with atmospheric pressure chemical ionization mass spectrometric method for quantitative determination of azithromycin in human serum; deuterium-labeled azithromycin was used as the internal standard (53).

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