High performance liquid chromatography (HPLC) of antibiotics of vancomycin type. Comparative studies.

Abstract

Comparative HPLC examination of seven antibiotics of vancomycin type has been undertaken. Investigation has shown that on column I in eluent system B, ristomycin A (ristocetin A) can be not only separated from vancomycin, but both antibiotics can be quantitatively determined. Under these conditions the lowest detectable quantities of the individual antibiotics have been also stated. By the application of this column and eluent system A, ristomycin A (ristocetin A) and the major component of the A-35512 B antibiotic complex can be readily separated from one-another and from avoparcin a and i.Recently the vancomycin group of antibiotics (vancomycin, actinoidin, ristocetin, ristomycin, avoparcin and antibiotic complex A-35512 B) have become both theoretically and practically important'.2).These antibiotics, except for vancomycin, are produced as mixtures of structurally related biologically active compounds. These glycopeptide-type antibiotics of relatively high molecular weight (1,420-2,063 daltons) often suffer decomposition upon the isolation process. Therefore the exact quantitation of the active factor in the antibiotic preparations is a very important task.The biological assay is restricted only to the determination of the total antibiotic activity of antibiotic preparations. Due to the similar chromophore system and specific optical rotation of the structurally related antibiotics and their decomposition products, ultraviolet spectroscopic3) and polarimetric') methods are suitable only in a limited number of cases and primarily for homogeneous preparations.For the detection of the vancomycin group of antibiotics and their inactivated products, paper') and thin-layer chromatographic') methods, as well as the combination of these procedures with bioautography have been used earlier with varying success. For the isolation and separation of the individual variants of the A 35512 antibiotic complex (novel members of the vancomycin-type antibiotics), MICHEL et at.') have first tried the application of HPLC by using reversed-phase ODS silica gel column and gradient elution technique. The purity of ristocetin-u-aglycones> was established by HPLC too, by means of reverse phase column (Spherisorb S 5 ODS) eluted with monobasic potassium phosphate -methanol (7: 3). According to ELLESTAD et al.9> avoparcin a and ;3 have been isolated and also separated by extensive preparative HPLC with Prep Pak-500/C19 cartridges for the solid support. Elution was carried out with a buffer solution consisting of 2.5 % acetic acid, 0.08 M ammonium hydroxide, 0.01 tit sodium heptane sulfonate, and containing of 13 -j 17 % acetonitrile.More recently MCCLAIN et a/.1O> have reported a HPLC method for the quantitative determination

Investigation of vancomycin and related substances by liquid chromatography/ion trap mass spectrometry

“…The mobile phases of the existing methods contain non‐volatile additives, e.g. phosphate24, 43, 45 or borate,34 or solvents that can give problems in combination with MS such as dioxane 46…”

“…Several methods have been used for the analysis of VM, such as thin‐layer chromatography (TLC),13 microbiological assay,14, 15 fluorescence polarization immunoassay,15–19 radioimmunoassay,15, 20 enzyme multiplied immunoassay,21 capillary electrophoresis (CE),22, 23 and liquid chromatography (LC) 24–46. Most of these methods were used to monitor VM concentration in blood.…”

“…allows the purity control of VM with micellar electrokinetic capillary chromatography (MECC). Only a few LC methods have been described for impurity testing of VM,24, 34, 43, 46 and there is an official LC method in the European Pharmacopoeia (Ph. Eur.…”