Aminoglycosides derivatization

Although a number of chromatographic methods have been reported for determinations of aminoglycoside antibiotics in blood serum and urine, the application of chromatographic methods to residue analysis has been very limited. Shaikh et al (93) recently described an HPLC method for neomycin in animal tissue, and Lachatre et al (94) described a method for nine aminoglycosides in plasma, urine, and renal cortex tissue. Both procedures use post column derivatization with a-pthalaldehyde and fluorescence detection. 

Trinitrobenzenesulfonic acid has been also used for the prechromato-graphic derivatization of aminoglycosides in alkaline media (228, 229). A major advantage of this label is the fact that hydroxyl groups cannot be concurrently derivatized, thus increasing, the selectivity. Unlike prementioned labels, 4-fluoro-... 

The separation mechanism in the LC analysis of aminoglycosides is usually highly dependent on the applied derivatization technique, either precolumn or postcolumn. This is due to the fact that a prerequisite of aminoglycosides analysis is most often suitable derivatization to produce fluorescent derivatives the presence of primary amine groups in most of the aminoglycoside antibiotics enables a number of derivatives to be readily formed. 

Aminoglycosides are basic, thermally labile, hydrophilic compounds that do not contain analytically useful UV-absorbing chromophores. Therefore pre- or postcolumn derivatization is necessary for their UV or fluorescence detection. 

Derivatization is the preferred option for aminoacids (making use of the anomalous ORD or CD effects introduced with the chromophore) to move the Cotton bands to longer wavelengths and to lower the limits of detection. Derivatizations might include N-acetylation as in the case of aminoglycosides [67], dansylation [72], or binding to metal complexes [73,74], see later. On the other hand aminoacids have an auxiliary role to play in the analysis of other substances, where advantage is taken of their chiroptical properties. 

Figure 18.9 tobramycin is an aminoglycoside. It was derivatized with o-phthalaldehyde in a 1.83 m X 0.38 mm i.d. capillary after passing through the column. Fluorescent products result from a combination of this reagent and amines after a reaction period of 6 s. 

The most frequently encountered mechanism of resistance to aminoglycosides is undoubtedly due to chemical derivatization of particular functional groups by R-factor-mediated enzymes, that is, coded by self-replicating extrachromosomal loops, or plasmids. This mechanism is primarily responsible for the spread of resistance and is therefore the one of most clinical significance. 

One innovation has been to derivatize the aminoglycosides by reaction with phenyl isocyanate (Fig. 7.8). Each aminoglycoside has multiple sites that can be derivatized. The reaction is facile, going rapidly to completion. The derivatives are better retained on reversed phase columns than the parent compounds and respond well to LC-MS analysis. This procedure has been used for the determination of a range of aminoglycosides in bovine milk down to concentrations of 10-12 pg/1. Weak cation exchange... 

Kim B-H, Lee SC, Lee HJ, Ok JH, reversed phase liquid chromatographic method for the analysis of aminoglycoside antibiotics using pre-column derivatization with phenyliso-cyanate, Biomed. Chromatogr. 2003 17 396-403. 

Turnipseed SB, Clark SB, Karbiwnyk CM, et al.. Analysis of aminoglycoside residues in bovine milk by liquid chromatography electrospray ion trap mass spectrometry after derivatization with phenyl isocyanate, J. Chromatogr. B 2009 877 1487-1493. 

Recent advances in the synthesis of aminoglycoside antibiotics have been reviewed. Derivatized dihydrostreptomycin has been hydrolysed to give a chiral streptidine derivative (9), which could be condensed with a dihydrostrepto-biosaminyl chloride derivative to give a- and j3-glycosides, the a-form yielding dihydrostreptomycin again on deprotection. ... 

Pyridine has also been used as the basic catalyst and chloride acceptor for benzoylation reactions. In one procedure, amines, volatile alcohols or thiols isolated by benzene extraction, are benzoylated with pyridine (1 ml) and benzoyl chloride (0.5 ml) by shaking intermittently at room temperature for several hours. The pyridine phase is extracted with 2 M HCl and the excess benzoyl chloride is hydrolysed with water for 12 hours. After shaking with 2 M sodium carbonate to remove benzoic acid, the benzene solution is dried and concentrated for analysis [144]. Aminoglycoside antibiotics are derivatized to the benzoyl derivatives in a similar reaction using 90 fi of pyridine and 10 /il of benzoyl chloride at 80 °C for 30 minutes. The pyridine is evaporated in a stream of nitrogen and excess benzoyl chloride is converted to methyl benzoate with methanol, again at 80°C, for 10 minutes. The product is cleaned up for analysis by a rather involved solvent extraction procedure [145]. 

Nitrobenzenes are most commonly used for the derivatization of amino compounds. The 4-nitrobenzoyl group has been shown to impart high UV absorptivity to amines. l-Fluoro-2,4-dinitrobenzene has been used as a label for aminoglycosides (Figure 5) such as neomycin [25, 26], fortimicin A [27], amikacin [28, 29], tobramycin [30-33], gentamicin and sissomicin [31-33]. 

A typical example of FDNB derivatization is the determination of amikacin in human serum [28,29], In a pre-column derivatization method, the aminoglycoside was extracted from serum by using a cation exchange column [29]. The eluate was treated with FDNB in dimethyl sulphoxide (DMSO) at pH 10. The derivative was then separated on an RP-Cjg column with water/ acetonitrile as mobile phase and monitored at 365 nm. The recovery of amikacin was only 72%, and kanamycin was used as an internal standard. The sensitivity was 1 mg 1 and 200 1 of serum was needed. 

Automated pre-column deiivadzation and h.p.l.c. analyses of aminoglycoside antibiotics have been reported, in an attempt to overcome problems associated with the instability of the derivatives. Reaction of amikacin with o-phthalaldehyde and mercaptoethanol in a borate buffer, reversed-phase separation, and u.v. detection at 340 nm produced two peaks, one from incomplete derivatization. It was claimed that the method was successfully applied to other aminoglycoside antibiotics containing a primary amino-group, i.e. gentamycin, tobramycin and kanamycin. Other workers have reported related derivatization and analysis conditions for tobramycin wherein mainly one peak was observed, though of unknown identify. ... 

Amikacin can also be determined by reversed-phase h.p.Lc. following derivatization with 1-fluoro-2,4-dinitrobenzene. The aminoglycoside antibiotics spectinomycin, hygromycin, streptomycin and dihydrostreptomycin were determined in bovine tissues by ion-pair reversed-phase h.p.l.c. using volatile ion-pair reagents, and by HPAEC. ... 

The following antibiotics have been determined the aminoglycoside tobramycin by chromatography on a pellicular anion-exchange resin, post-colunm addition of alkali, and pulsed amperometric detection pseudouridine with S-fluorouridine as an internal standard on a reversed-phase column with post-column periodate oxidation, derivatization with meso-... 

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