High performance liquid chromatography of chlortetracycline and related substances on poly(styrenedivinylbenzene)copolymer

Summary An isocratic high performance liquid chromatographic method has been developed for assay and purity control of chlortetracycline, which is separated from all related compounds. The stationary phase is poly(styrene-divinylbenzene)copolymer (PSDVB), which is heated at 60°C. The mobile phase is 2-methyl-2-propanol-1.0 M perchloric acid (5.0 ml)-water (up to 100 ml). The amount of alcohol modifier varies between 2.5 and 6.5% m/v depending upon the brand and porosity of PSDVB used. The flow rate is 1.0 ml/min and UV detection is at 254 nm. The total analysis time does not exceed 40 min. Although the method has been shown to be applicable with several brands of 100 Å PSDVB e.g. PLRP-S, PRP-1, RoGeL and TSK-Gel, the best separations were obtained on the wide pore material PLRP-S 100 Å.     

Liquid chromatography of polymyxin B sulphate

A reversed-phase liquid chromatography method for analysis of polymyxin B sulphate is described. The method uses a YMC-Pack Pro, C₁₈, 5 μm, 250×4.6 mm I.D. column maintained at 30°C. The mobile phase comprises acetonitrile–sodium sulphate (0.7%, m/v)–phosphoric acid (6.8%, v/v dilution of 85%, m/m phosphoric acid)–water (22.25:50:5:22.75) at a flow-rate of 1.0 ml/min. Detection was by UV at 215 nm. The method is able to resolve polymyxin B₁, the major component, from more than thirty other components present in the complex. Robustness was evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity.     

Liquid chromatographic method for analysis of saponins in Maesa balansae extract active against leishmaniasis

A liquid chromatographic method was developed for the separation of six related triterpenoid saponins in Maesa balansae extracts with different purity, active against leishmaniasis. As stationary phase a Hypersil BDS C18 column (3 microm), 100 x 4.6 mm was used. The mobile phase was a mixture of methanol, acetonitrile, 5% (m/v) ammonium acetate, pH 6.5 and water. A linear gradient was developed for the analysis of crude extracts. An isocratic method was developed to analyze purified samples that mainly contained saponins 3 and 4, the most active saponins. The isocratic LC method was optimized and the robustness was evaluated with an experimental design. The method showed good selectivity, repeatability, linearity and sensitivity.     

Liquid chromatographic separation of monoamino analogues of dideoxyadenosine and 9-(dideoxy-β-D-lyxo-pentofuranosyl)adenine from adenine

Summary A liquid chromatographic method that can separate each of the monoamino analogues of dideoxyadenosine and 9-(-dideoxy--D-lyxo-pentofuranosyl)adenine from their main degradation product, adenine, is described. The influence of the pH of the mobile phase, the type and concentration of the organic modifier and the concentration of the silanol masking agent and the buffer have been investigated. Several reversed phases were examined. Samples were finally analyzed on a 10 m Spherisorb ODS1 column (250 mm×4.6 mm I.D.) using acetonitrile –0.2 M potassium phosphate buffer pH 6.0–0.2 M tetramethylammonium phosphate pH 6.0-water (550.589.5, v/v) as the mobile phase. The interactions between the solute and the stationary phase are discussed in view of the basic properties of the compounds under study.     

毛细管电泳法分离四环素及其杂质

用毛细管电泳方法分离四环素和它的主要杂质４－差向四环素，脱水四环素，４－差向脱水四环素和２－乙酰基－２－脱酰胺四环素，相对次要的杂质氯四环素和脱甲四环素也能被分 离。     

Isolation of doxycycline, 6-epidoxycycline and 2-acetyl-2- decarboxamidometacycline from commercial metacycline by preparative column liquid chromatography on silica gel.

Isolation of doxycycline, 6-epidoxycycline and 2-acetyl-2-decaboxamidometacycline from commercial metacycline was achieved by preparative column liquid chromatography on silica gel, previously impregnated with edetate (EDTA). Careful control of the pH of EDTA allowed fine tuning of the separation. The mobile phases were composed of dichloromethane, methanol and .1 mM EDTA at pH 9.0 or 6.0. Structures were confirmed with nuclear magnetic resonance spectroscopy. The presence of doxycycline and its 6-epimer in commercial metacycline has not previously been described. The presence of the 2-acetyl derivative was not surprising since analogous 2-acetyl derivatives have been identified in other tetracyclines.     

Evaluation of silanol-deactivated silica-based reversed phases for liquid chromatography of erythromycin

The suitability of eleven silanol-deactivated reversed phases for the liquid chromatography of erythromycin was investigated. The selectivity and efficiency of each stationary phase were examined. The performance was compared to that of a non-deactivated C₁₈ silica-based reversed-phase material, Hypersil C₁₈ (5 μm). Two types of mobile phases were used, one containing no tetrabutylammonium (TBA) and the other containing TBA. Addition of TBA as a silanol-blocking agent improved the theoretical plate number and symmetry factor of the peaks corresponding to erythromycin A (EA) and erythromycin A enol ether for all the deactivated reversed phases. These results are an indication of the presence of some residual silanol activity in these phases. Separation of erythromycin E and EA was achieved on only two of the eleven phases. The selectivity was always poorer than that obtained in a previously described method using a poly(styrene-divinylbenzene) stationary phase.     

Separation of erythromycin and related substances on base-deactivated reversed-phase silica gel columns

An official liquid chromatographic method for the analysis of erythromycin and related substances, which is based on a polymer reversed-phase, is described in the European Pharmacopoeia and in the United States Pharmacopeia. The pH of the mobile phase used in this system is 9.0. Recent advanced technology has led to the introduction of a new generation of silica-based reversed-phase column packings, which are claimed to be much more stable towards bases. They are useful for the analysis of basic compounds. Studies to verify the separation of erythromycin and related substances on Hypersil BDS C₁₈, Luna C₁₈(2), Inertsil ODS-2 and Supelcosil ABZ+ have been performed and the results are presented. It is shown that these base-deactivated phases give a better sensitivity and selectivity towards erythromycins than the polymer phase, provided that an adapted mobile phase is used. This is the first liquid chromatographic method described for the separation of erythromycin D from erythromycin A.