Cleaning validation and using Ethanol instead Acetonitril

[marina1111]

Hello!
I have 2 questions, and I am not sure the first one is legitimate, so if it is not please tell me.

1) How should look sample sequence for cleaning validation? How many injection of SST? Should the SST be the limit concentrations? How many concentrations should be used? I use now 7 concentrations – 3 around the limit concentration, 2 lower, LOQ and LOD, and use 2 calibration curves for higher and lower concentrations – is that logical? Can I find guidelines anywhere?

2) Can be EtOH considered as substituent to Acetonitrile? Are there any disadvantages in addition to high viscosity (such as shorter column lifetime)?

Thank you very much!

[Consumer Products Guy]

2) Can be EtOH considered as substituent to Acetonitrile? Are there any disadvantages in addition to high viscosity (such as shorter column lifetime)?

HPLC grade ethanol or methanol can be used in many cases to substitute for ACN. Yes, pressure will go up, and you'll likely need to increase temperature to help with that, and you'll need to use a higher percentage of organic. But substutuing for ACN is not usually a drop-in, especially if you have other components which you need to resolve from you analyte. See my post under "Acetonitrile shortage" where I describe the analyte eluting differently in comparison to fragrance component peaks in a finished product using methanol. And if your ACN test procedure is a validated/regulated procedure, then it looks like you've got a revalidation coming after you solve the technical issues. No, ethanol or methanol will not affect column life.

[marina1111]

Thank you for the answer, our methods are not validated yet, and we are trying to substitute for relatively easy separations such as dissolution

[Jade.Barker]

Sorry if this doesn't apply, I am pretty new to HPLC... but I worked with ethanol a lot in my last lab as a sample component.

I would be careful about with what compenents you use it with, Ethanol is great at turning into other things! We were doing Ethanol Fermentation Samples (Corn Mash). Samples with high Lactic Acid and Ethanol can start to get bad resolution because once it is together they can make Ethyl Lactate. Also, I had a sneaky feeling that High amounts of Ethanol were acting as a slight acid. I know it can do it "in theory", I just didn't have time to prove it. It is possible that the Glucose peak was getting some acid related shouldering.

Just my 2 cents. Good Luck!

[HW Mueller]

On the acidity of ethanol:
Some dissociation constants (autoprotolysis),
H2O = 1x10^-14
MeOH = 2x19^-17
EtOH = 8x10^-20

I wouldn´t expect (and havn´t seen it either) EtOH to be particularily reactive in the HPLC context.

[marina1111]

Thank you!

[krickos]

Hi

Question 1. Well §12.7 in ICH Q7a gives some information about cleaning in API production but not regarding SSTs in cleaning methods, assuming we are talking pharmaceutical requirements here.

Recall that we are typically talking trace methods here with sometimes more or less complicated sample treatment (extraction/evapouration/direct injection of swabb/cleaning fluids) so the typical found SSTs in assays and impurity testing may not always fit as good.

Hard to give a general suggestion as it also depends a bit on in which concentration area you work in, the response of your analyte, toxilogical/physiological activity of the compund etcetera. But I can share some SSTs I have seen lately in cleaning methods that seemed "scientificly sound":
Blanks
4-5 calibration levels-control of r-Square
Signal to noise check for lowest calibration solution
rsd 2-4% for the obtained responsfactors (or use an internal standard in calibration solutions to check RSD)
RT and/or N control of analyte in strongest calibration solution
recheck of signal to noise after last sample injection.

[marina1111]

Thank you so much! It is a great help!