求红霉素HPLC方法，关键是色谱条件。
谢谢
参考一下这篇文献吧。
改变检测波长HPLC法测定红霉素的含量.PDF(165.69k)
BP2001
Examine by liquid chromatography
Test solution. Dissolve 40.0 mg of the substance to be examined in a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS in a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (b). Dissolve 10.0 mg of erythromycin B CRS and 10.0 mg of erythromycin C CRS in a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 50.0 ml with the same mixture of solvents.
Reference solution (c). Dissolve 5 mg of N-demethylerythromycin A CRS in reference solution (b). Add 1.0 ml of reference solution (a) and dilute to 25 ml with reference solution (b).
Reference solution (d). Dilute 3.0 ml of reference solution (a) to 100.0 ml with a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R.
The test solution and reference solutions can be used within one day if stored at 5癈.
The chromatographic procedure may be carried out using:
?a column 0.25 m long and 4.6 mm in internal diameter packed with styrene-divinylbenzene copolymer R (8 祄 to 10 祄) with a pore size of 100 nm,
?as mobile phase at a flow rate of 2.0 ml per minute a solution prepared as follows: to 50 ml of a 35 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 with dilute phosphoric acid R, add 400 ml of water R, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to 1000 ml with water R,
?as detector a spectrophotometer set at 215 nm,
?a 100 祃 loop injector,
?an electronic integrator,
maintaining the column at 70癈, preferably using a water-bath. Inject reference solution (c). Adjust the sensitivity of the detector so that the height of the peaks is at least 25 per cent of the full scale of the recorder. The substances are eluted in the following order: N-demethylerythromycin A, erythromycin C, erythromycin A and erythromycin B. The test is not valid unless the resolution between the peaks corresponding to N-demethylerythromycin A and erythromycin C is at least 0.8 and the resolution between the peaks corresponding to N-demethylerythromycin A and erythromycin A is at least 5.5. If necessary, adjust the concentration of 2- methyl-2-propanol in the mobile phase or reduce the flow rate to 1.5 ml or 1.0 ml per minute. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of the peak area for erythromycin A is not greater than 2.0 per cent. Inject alternately the test solution and reference solutions (a) and (b).
Calculate the percentage content of erythromycin A using the chromatogram obtained with reference solution (a). Calculate the percentage contents of erythromycin B and erythromycin C using the chromatogram obtained with reference solution (b).
中国药典2005版公示版：
流动相为：0.2mol/L磷酸铵缓冲液（取磷酸二氢铵1.15g，加水50ml溶解，三乙胺调PH至6.5）-0.2mol/L四甲基氢氧化铵溶液（取25%四甲基氢氧化铵水溶液14.6ml，加水100ml，用磷酸调节PH至6.5，再加水稀释至200ml）-乙腈-水（5：20：30：45）
色谱柱：用十八烷基硅烷键合硅胶为填充剂（BDS柱，4.6x250mm）
检测波长：215nm
流速：1.0ml/min
系统试用性试验：精取红霉素对照品10mg，置10ml量瓶中，加甲醇5ml溶解后，用磷酸盐缓冲液（ph3.5）（取磷酸盐缓冲液（ph7.0）20ml，用磷酸溶液调节ph至3.5，即得）稀释至刻度，室温放置30分钟以上，取20微升注入HPLC，红霉素峰与其降解物红霉素烯醇醚峰的分离度应大于14。红霉素烯醇醚峰的保留时间为红霉素A的3.5倍。理论板数按红霉素峰计算应不小于1000，拖尾因子应小于2.5。
磷酸盐缓冲液（PH7.0）-甲醇（15：1），
中国药典2010版本的方法
1.色谱条件：
色谱柱：C18,4.6*250mm*5um
波长：215nm；流速：0.8~1.0ml/min，柱温35℃，进样量20ul
2.流动相：
磷酸盐溶液（取磷酸氢二钾8.7g，加水1000ml，用20%磷酸调节PH值至8.2）：乙腈（40：60）为流动相；
3.稀释剂溶液： 磷酸盐缓冲液PH=7.0（称取磷酸二氢钠﹒2H2O1.22g，磷酸氢二钠﹒12H2O 4.37g加水100ml，调节PH=7.0）----甲醇(15:1)
用于做发酵液分离效果不是很理想，不知哪位高手有更好的方法
晕，这个是我2004年发的帖子啊，怎么又被发出来了，呵呵
楼上找茬的，2004年都做过了还问。
谢谢
参考一下这篇文献吧。
改变检测波长HPLC法测定红霉素的含量.PDF(165.69k)BP2001
Examine by liquid chromatography
Test solution. Dissolve 40.0 mg of the substance to be examined in a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (a). Dissolve 40.0 mg of erythromycin A CRS in a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with the same mixture of solvents.
Reference solution (b). Dissolve 10.0 mg of erythromycin B CRS and 10.0 mg of erythromycin C CRS in a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R and dilute to 50.0 ml with the same mixture of solvents.
Reference solution (c). Dissolve 5 mg of N-demethylerythromycin A CRS in reference solution (b). Add 1.0 ml of reference solution (a) and dilute to 25 ml with reference solution (b).
Reference solution (d). Dilute 3.0 ml of reference solution (a) to 100.0 ml with a mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R.
The test solution and reference solutions can be used within one day if stored at 5癈.
The chromatographic procedure may be carried out using:
?a column 0.25 m long and 4.6 mm in internal diameter packed with styrene-divinylbenzene copolymer R (8 祄 to 10 祄) with a pore size of 100 nm,
?as mobile phase at a flow rate of 2.0 ml per minute a solution prepared as follows: to 50 ml of a 35 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 with dilute phosphoric acid R, add 400 ml of water R, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to 1000 ml with water R,
?as detector a spectrophotometer set at 215 nm,
?a 100 祃 loop injector,
?an electronic integrator,
maintaining the column at 70癈, preferably using a water-bath. Inject reference solution (c). Adjust the sensitivity of the detector so that the height of the peaks is at least 25 per cent of the full scale of the recorder. The substances are eluted in the following order: N-demethylerythromycin A, erythromycin C, erythromycin A and erythromycin B. The test is not valid unless the resolution between the peaks corresponding to N-demethylerythromycin A and erythromycin C is at least 0.8 and the resolution between the peaks corresponding to N-demethylerythromycin A and erythromycin A is at least 5.5. If necessary, adjust the concentration of 2- methyl-2-propanol in the mobile phase or reduce the flow rate to 1.5 ml or 1.0 ml per minute. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of the peak area for erythromycin A is not greater than 2.0 per cent. Inject alternately the test solution and reference solutions (a) and (b).
Calculate the percentage content of erythromycin A using the chromatogram obtained with reference solution (a). Calculate the percentage contents of erythromycin B and erythromycin C using the chromatogram obtained with reference solution (b).
中国药典2005版公示版：
流动相为：0.2mol/L磷酸铵缓冲液（取磷酸二氢铵1.15g，加水50ml溶解，三乙胺调PH至6.5）-0.2mol/L四甲基氢氧化铵溶液（取25%四甲基氢氧化铵水溶液14.6ml，加水100ml，用磷酸调节PH至6.5，再加水稀释至200ml）-乙腈-水（5：20：30：45）
色谱柱：用十八烷基硅烷键合硅胶为填充剂（BDS柱，4.6x250mm）
检测波长：215nm
流速：1.0ml/min
系统试用性试验：精取红霉素对照品10mg，置10ml量瓶中，加甲醇5ml溶解后，用磷酸盐缓冲液（ph3.5）（取磷酸盐缓冲液（ph7.0）20ml，用磷酸溶液调节ph至3.5，即得）稀释至刻度，室温放置30分钟以上，取20微升注入HPLC，红霉素峰与其降解物红霉素烯醇醚峰的分离度应大于14。红霉素烯醇醚峰的保留时间为红霉素A的3.5倍。理论板数按红霉素峰计算应不小于1000，拖尾因子应小于2.5。
磷酸盐缓冲液（PH7.0）-甲醇（15：1），
中国药典2010版本的方法
1.色谱条件：
色谱柱：C18,4.6*250mm*5um
波长：215nm；流速：0.8~1.0ml/min，柱温35℃，进样量20ul
2.流动相：
磷酸盐溶液（取磷酸氢二钾8.7g，加水1000ml，用20%磷酸调节PH值至8.2）：乙腈（40：60）为流动相；
3.稀释剂溶液： 磷酸盐缓冲液PH=7.0（称取磷酸二氢钠﹒2H2O1.22g，磷酸氢二钠﹒12H2O 4.37g加水100ml，调节PH=7.0）----甲醇(15:1)
用于做发酵液分离效果不是很理想，不知哪位高手有更好的方法
晕，这个是我2004年发的帖子啊，怎么又被发出来了，呵呵
楼上找茬的，2004年都做过了还问。