Method development

[brasileo_88]

I was interessted in determining the assay and related substances of spectinomycin by HPLC. I know about a method that uses ECD detector.
I intented on using other method because i haven't got the ECD detector. The method consist of a normal phase and using a derivatization agent. Does any one know if this method is useful?

The normal phase method using UV Detection was taked from Chromatography journal 333.
Thanks

brasileo@gmail.com

[Fernando]

Hi brasileo_88

Consult this article.

A Validated Stability-Indicating HPLC Method for Routine Analysis of an Injectable Lincomycin and Spectinomycin Formulation

Murad N. Abualhasan,*,1 Nidal Batrawi,2 Oliver B. Sutcliffe,3 and Abdel Naser Zaid1

Best luck

Fernando

[brasileo_88]

Thanks Fernando
I'll try

[artsjeroen]

I don't know what is in your sample matrix, but if the selectivity of ECD is not necessary I would go for HPLC UV.

We measure the activity of Spectinomycin using reversed phase on C18 with UV detection (215 nm).
Using SDS in the mobile phase for ion pairing. Works well for us, with good symmetrical peaks.

[brasileo_88]

Thanks for your answer.

It is listen good, may you have operating condition for you method?
It is valid for related substances too?

Thanks

[artsjeroen]

60% - 2 g SDS + 15 ml H3PO4 in 1000 ml water (pH = 6.0 with Ammonia).
40% - Acetonitrile

flow = 1 ml/min
T = 35°C
Column is symmetry C18, 5 µm, 3.9 x 150 mm, but I guess any C18 with comparable dimensions will work.

Our solutions are the raw material dissolved in water (ca. 2.5 mg/ml).
We don't measure related substances but you could give it a try.

Please let me know if it works for you.

[brasileo_88]

Thanks but your method can not be use to deremined related subst.

[artsjeroen]

Did it work for assay determination? What was the retention time of Spectinomycin? And how was the peak shape?

With altering column length/mobile phase I think you can get more resolution on the chromatography of the related substances, I don't know why it would not work.