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Sorry in advance for the long winded explanation...
I'm trying to separate about 12 known impurities from a two component drug product. After some method development I decided on developing a gradient method using ion pairing agents. Below is the full chromatographic condiitions.
Everything started going well during validation, multiple columns and multiple systems were reproducible. Then all of a sudden we started injecting LOQand resolution and we were getting a TON of noise making integration difficult.
Now I have a very concentrated solution of one active and reasonable concentration of the other (65,000ug/ml compound a, 500ug/ml compound b). Consequently, the placebo load as you could imagine is quite high.
I've heard that making gradients with odsa Is not recommended however I didn't have much of a choice. I needed enhanced selectivity and the main component and impurities are ketones and according to what I've found online exhibit extreme fronting due to gem diol formation and hemiketal formation.
I also noticed that when sample was injected prior to system suitability that the noise seemed to reduce. I tried making 3 quick injections of selectivity soliton followed by the normal gradient and then followed by 1 more injection of selectivity and then start into my system suit. When I did this however I am only seeing solvent front and component a, then no other peaks. Noise seems to have been reduced but again I don't see anything except solvent front and gradient baseline shift
1. Out of general curiosity, was my approach towards making a gradient method good? The idea was to as accurately as I could get the SAME concentration of ODSA in mpa and mpb? I was noticing when I first was playing around with this that I was getting some baseline noise but it was tolerable but then recently something happened and now I peaks are masked by an extremely noisy baseline.
2. In my attempt to "condition" the column, could I have gone overboard and that's why I'm seeing no peaks (other than the 65,000ug/ml component)?
3. As a side note, during the development we had also observed that different manufacturers of ODSA gave slightly different selectivity. This was confirmed by a colleague of mine and I was wondering if anyone had any ideas about why this is? We ended up during validation to stick with the same manufacturer of ODSA, and had opened a few different bottles from that same manifacturer but then we opened up smoke new one and we started observing the baseline noises.
Stock buffer solution: 10grams octanesulfonic acid, 2.5 grams potassium phosphate mono basic , 4ml tea per liter ph 3.0
Mobile phase a: 460ml buffer: 500ml water: 40ml acn
Mobile phase b: 460ml buffer:40ml water:500ml acn
Chromatographic conditions:
Injection volume 10ul
Flow:1.5ml/min
Temperature:50c
Column: Luna c18(2) 100x4.6, 5um
Wavelength: 235nm
Gradient:
0->15 minutes 100%a
15->16minutes 93:7
16->25minutes hold 93:7
25->45 50:50
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