Improved methods for the separation and quantitation of cephalosporins, penicillins, tetracyclines and several miscellaneous antibiotics by reverse phase high speed liquid chromatography are presented. The methods have been improved significantly by the substitution of high efficiency, small particle (-10 pm) reverse phase columns in place of the previously used medium efficiency, pellicular columns. The conditions and procedures described here illustrate that considerable improvements in separation and sensitivity of detection of antibiotics are achieved. Pure compounds, complex mixtures of antibiotics in a variety of dosage forms and fermentation broths are routinely analyzed by the described procedures.In a previous paper" we described conditions for the determination of cephalosporins, penicillins, tetracyclines and other miscellaneous antibiotics by reverse phase high speed liquid chromatography.A second paper 2) dealt with a specific application of this technique to the determination of cephalexin and cephradine in various pharmaceutical formulations and in physiological fluids.

In the first papers' of this series we described conditions for the determination of cephalosporins, penicillins, tetracyclines and other miscellaneous antibiotics by reverse phase high speed liquid chromatography. In the second paper 2 we presented improved methods which use high efficiency, small particle (10 /gym) reverse phase columns for the determination of these compounds. The application of reverse phase methods to the analysis of cephalosporins and penicillins has also been reported by other investigators'`''.A review article which discusses penicillins and cephalosporins as well as other types of antibiotics and drugs has also been published'). Cephalexin and cephradine have been determined in plasma and urine by reverse phase methods","). Penicillin and cephalosporin diastereoisomers have been separated by reverse phase HPLCs2•s3'. Also, a normal phase methodl4•s~) which employs an amino column has been reported for the analysis of cephalosporin C derivatives and cephalosporin antibiotics.For reasons such as discussed in our first papers), we prefer to use reverse phase instead of ion exchange chromatography for antibiotics. However, ion exchange high pressure liquid chromatography has been used successfully by other investigators for the analysis of cephalosporins1°,17,1s) and penicillins1° '° . Most of the methods reported in our first papers' as well as much of the work reported by other investigators used large particle (-37-50 pcm), medium efficiency pellicular columns. These columns are adequate for many applications, but there are many disadvantages such as poor separation, long analysis times, poor sensitivity and low sample capacity. The use of high efficiency, totally porous, small particle (-10 /um) reverse phase columns as reported in our second paper') overcame most of these disadvantages and gave significant improvements in performance. The primary advantages associated with these columns are: (1) somewhat better separations; (2) shorter analysis times;(3) sharper peaks; (4) higher sensitivity; (5) greater retentivity and (6) larger sample capacity.