Recovery Problems

[Jana P]

Hi All

I am currently running a method to detect residual methanol and ethanol in one of our products. I am having an issue with getting 100% recovery from my samples.

I am running on an Agilent 6890NGC and G1888 Headspace sampler.

When I analyse a spiked drug substance sample on its own I obtain 100% recovery for ethanol and methanol. When I analyse a spiked placebo sample (i.e. product excipients minus drug) I obtain 100% recovery. However when I analyse a spiked solution containing drug and placebo my recovery jumps up to 120% for ethanol and 105% for methanol.

I am a little lost as to what might be happening in the drug+placebo sample that would cause the recovery to increase by 20%.

There is no evidence of interfering peaks contributing from the drug substance or the placebo mixture.

Could the pressure build up in the vial be a contributing factor? As in with drug substance and placebo in the vial the pressure is greater?? I am really at a loss and a little over my head with coming up with an explanation.

Any ideas or suggestions would be appreciated.

Thanks all

[krickos]

Hi Jana

First, well it is quite common that you do not obtain a 100% recovery in residual solvent analysis, especially with a headspace injection. One is is usually happy with a 90-110% recovery but 80-120% tends also to be accepteble.

As you are in Australia I presume you know that APVMA is one of few (if any else) agencies that actually stated expected recovery intervals in their validation guideline (october 2004):
0,1-1 % 80-120% recovery
<0,1% 75-125% recovery

A few question:
No interfering peak from mixture of placebo and drug substance? (not just the individual ones)

How much of each solvent did you actually put into the vial? (ie total amount in vial not concentration versus product weight)

One teorectical thought:
Assuming you dissolve in water, the most polar alkohol is most sensitive to "salts" (salting out effect) ie methanol would be more likely to increase if the is some salt formation when mixing drug substance and placebo.

This relation is reversed when incresing water content in DMF from lets say 10% to 20% ie the least polar alkohol will increase most in area, so for example water formation due to drug substance/placebo interaction in a DMF solution can cause matrix effects.

That brings me to the last part, if your recovery is not accepteble to your standards due to matrix effects using external standards you have to shift to the standard addition technique instead.

[Jana P]

Hi Krickos

We are not satisfied with a recovery of as high as 120%, if it was in the 110% range it is something we could live with.

There are no interfering peaks from the drug substance or the placebo mixture.

I think that the problem is due to the salting out effect. I have re-analysed the drug substance on its own and the drug substance spiked. The recovery of ethanol is 125% and methanol is 115%. Thus I believe that it is the drug substance itself which is casing the salting out effect. Just to ensure my understanding of what is occurring in the sample, the presence of an electrolyte/salt in my solution is causing the solvent (in my case ethanol and methanol) to be more concentrated in the gaseous part of the headspace vial (The partition coefficient has changed?)

I appreciate your time and advice, Thank you.

[krickos]

Just to ensure my understanding of what is occurring in the sample, the presence of an electrolyte/salt in my solution is causing the solvent (in my case ethanol and methanol) to be more concentrated in the gaseous part of the headspace vial (The partition coefficient has changed?)

Hi Jana

Exactly, this is what can happen when analysing "salt" (sodium, magnesium etc) drug substances.
You have not stated what drug substance or sample solvent you use, but if you use water to dissolve the sample you might be able to work around this (ie avoiding standard addition) by adding lets say 0,25g Na2SO4 to all vials.
Hopefully the sodium sulphate will thus give an equal salting out effect in both external standards and sample and recoveries should be closer to 100%.

Books like "Static headspace-gas chromatography, theory and Practice" by Bruno Kolb and Leslie S Ettre usually cover "salting out" effects from a perspective that you can improve the partition coefficient on polar compounds, but rarely in the same section discuss that your sample can cause an unwanted one.

[kalidassa]

Hai
May i know the solvent u have used. u can try to get good recovery result by using the solvent DMF: WATER in the ratio of 90:10, 80:20 ect.,



Kalidass

[krickos]

Hai
May i know the solvent u have used. u can try to get good recovery result by using the solvent DMF: WATER in the ratio of 90:10, 80:20 ect.,

Kalidass

Hi Kalidass

Intresting, have you managed to reduce "salting out" effects by this approach?

I am not saying I wont work (might be sample depended and is of course sample amount dependant) but I have got >110% recoveries of sodium drug substances salts with regard to methanol in pure DMF (about 0,5g sample in 4-5ml DMF).

[kalidassa]

Hai
we have done lot of validations based on these combination of DMF: Water, we have used 50:50 also. adding buffer salts is one of the method to achive good recovery in HSGC. But we want to use high grade Buffer salts to avoid unknown peaks or degradation peaks produced by that buffers during the analysis. iIn furture u can try with these combinations

Best wishes

Kalidass

[Jana P]

Hi All

Thank you for all your comments and advice.

I have managed to correct my recovery by playing around to find the correct amount of Sodium Salt (Na2SO4 as suggested by Krickos) to add to my standard preparations to give a recovery of approximately 100%.

I can now add this amount to my standard every time and my recoveries are perfect.

Thank you again for helping me to understand the GC process better.

[aldehyde]

Well you know where to look next time you get stumped, glad to hear that they fixed your problem!