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Gentamicin |
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Introduction Gentamicin is an antibiotic administered to patients suffering from potentially life-threatening bacterial infections. It has a narrow therapeutic range and constant monitoring is necessary due to the fact that excess dosage can cause kidney and auditory nerve damage. Chemically, gentamicin is exceptionally stable, and is used extensively in animal husbandry. It can be stored at elevated temperatures for extended periods-of-time without loss of biological activity. Gentamicin occurs in four optically active analogs as pictured here.
Because of its stability and antibiotic activity, it is used in dairy cattle, but treated animals should be held from production until the antibiotic clears their system (~ 2 weeks). Unfortunately, this has not always been the case and gentamicin residues have been detected in commercially produced-milk and milk products. It would obviously be useful to develop a sensitive method for detecting these materials, allowing subsequent identification of the source of the contamination in order to minimize public exposure. Gentamicin Lacks a Chromophore The problem of detecting gentamicin is compounded by the fact that it does not absorb in the UV or visible region of the spectrum. Unfortunately derivatization, to enhance detection, eliminates the subtle chemical differences between the four analogs making chromatographic separation impractical. However, gentamicin’s optical activity not only offers an opportunity to differentiate between the four analogs but also overcomes the absence of a good chromophore. Detection via Laser Polarimetry The figure below shows an analysis of gentamicin analogs from two different commercial suppliers using the PDR-Chiral Advanced Laser Polarimeter. The separation was accomplished using reverse phase, ion pair chromatography. The eluent consisted of methanol and 0.4 M TFA/H2O (80:20, v/v) with a flow rate of 0.75 mL/min. The total injected amount was 10 mg.
This is the first demonstrated separation of the four gentamicin analogs in a single separation system. The polarimetric detection system can detect gentamicin below the 50-ng level without requiring derivatization. Previous work used UV/Vis detection with derivatization but the added moiety dominated the chromatographic properties of the derivatives making it impossible to separate all four analogs. Note that with all four analogs separated, differences are clearly observed in the distribution of the four components from samples obtained from the two commercial suppliers. Identification of the source of gentamicin involved in a contamination is now possible. Specific Rotation of Gentamicin AnalogsPreviously, only the specific rotation of the enantiomeric mixture was reported due to the difficulty in separating and detecting all four analogs. However, with all four gentamicin analogs separated, as shown above, the polarimeter can be used to determine the specific rotation of each of the four analogs. First, the relative masses for each enantiomer were apportioned in a separate experiment using a refractive index detector. Using the measured optical rotations and the relative abundance of each of the four analogs (table below), a calculated value for the mixture was obtained. This calculated value (for the mixture) agrees with the accepted value found in the literature. Structurally, the four are virtually identical. However, the specific rotations are clearly distinct, underscoring both the selectivity of the polarimetric detector and the sensitivity of specific rotation to the arrangement of atoms at or near the chiral center.
A Practical Application In order to screen a large quantity of milk samples for possible contamination, a method must be both sensitive and rapid. The selectivity of the polarimeter is a distinct advantage in such applications. For this protocol, the milk sample was acidified with 0.1M trifluoracetic acid, centrifuged for 5 minutes, and then the supernatant was directly injected into the HPLC. The reverse phase, ion pair separation was identical to that used above. UV/Vis detection showed that a large number of compounds were present in the milk extract. However, most of these are not optically active and thus do not produce a response with the polarimeter, the exception being a distribution of material eluting just after 2 min.
With the polarimetric detector (see above figure), the gentamicin contamination was easily observed and the supplier of the gentamicin-contaminated milk sample was easily identified. The entire analysis took less than 15 minutes, which is a significant improvement over the extraction/cleanup/derivatization protocol used previously with UV/Vis detection. The UV/Vis protocol typically requires several hours precluding the possibility of its use in the widespread screening of milk samples. | |||||||||||||||||||||||||
