An isocratic reversed-phase high-pressure liquid chromatographic method for the determination of six components of the teicoplanin complex in biological fluid was developed. By using fluorescence detection after precolumn derivatization with fluorescamine, the assay is specific and highly sensitive, with reproducibility studies yielding coefficients of variation ranging from 1.5 to 8.5% (at 5 to 80 ,ug/ml). Response was linear from 2.5 to 80 ,ug/ml (r = 0.999); the recovery from spiked human serum was 76%. An external quality control was performed to compare this high-pressure liquid chromatographic method (H) with a standard microbiological assay (M); no significant deviation from slope = 1 and intercept = 0 was found by regression analysis (H = 1.03M -0.45; n = 15).Teicoplanin is a new glycopeptide antibiotic produced by Actinoplanes teichomyceticlls (1, 10) which shows appreciable activity against staphylococci and streptococci (9, 13), corynebacteria (7), listeria (8), and clostridia (Y. Glupczynski, M. Labbe, F. Orokaert, and E. Yourassowsky, Letter, Eur. J. Clin. Microbiol. 3:50-51, 1984). It appears to inhibit cell wall biosynthesis in gram-positive organisms by interfering with peptidoglycan polymerization (12). Pharmacokinetic investigations showed a multiexponential concentration in blood-versus-time profile with elimination halflives of 45 to 50 h (13), which is 6 to 10 times longer than that of vancomycin. In a recent review, Williams and Gruneberg (14) reported on the in vitro and in vivo activity, pharmacology, and toxicology of teicoplanin. They recommended that levels in serum be monitored until the safety of this potentially valuable antibiotic is assured. Furthermore, they recommended additional studies to examine penetration of teicoplanin into various body fluids and to assess its therapeutic efficacy. Toxicity must be avoided in ongoing clinical trials by reasonable dosage recommendations formulated on the basis of concentrations in serum. Consequently, an accurate, sensitive, and specific assay for the determination of teicoplanin is needed.The development of a high-pressure liquid chromatographic (HPLC) assay is difficult, since teicoplanin is a mixture of several related substances. The structures of its main components, A2-1, A2-2, A2-3, A2-4, and A2-5, and of the pseudoaglycones, A3-1 and A3-2, have recently been elucidated (2, 3, 6). Several minor constituents, designated A2-la, A2-lb, A2-1c, A2-ld, A2-le, A2-3a, and A2-5a, were also detectable by use