Separating vitamin D2 and D3 in RP column

[zheyin]

for stereo isomers, going to very low temperature most of the time improves the separation
we are talking going to 5-10 degrees set in the cooler
i wonder, if not switching the column is because of budget reason, then remember that ACN costs about 3 times more than MeOH.
so actually buying another column instead of switching between the 2 solvents can be cheaper over time, and the more the application is used, the faster the column repays itself.

Most management won't think in your such logical way,
since ACN is already there sitting in the lab
But a new column takes $$$ to receive.

[unmgvar]

zehin
in most labs budget spending defies logic. and it is also defined by 2 train of thoughts:
1. regulation locks you.
2. in the end you will pay what it takes to get the job done.

i know a company lab that works only with USP and EP standards and they do not use working standards.
a lot cheaper, but it requires them to do some paper work that they find to be too much work and time wasting.
before the ACN crisis we had a trend to move all dissolution application to HPLC. then when prices rocketed, we saw that it was cheaper to keep developing water based UV spectro methods where we could, and look at in-situ solution again for automation. but we are now locked with all those HPLC methods for a few years.

also because budgets are divided into several categories, labs tend not to see how much they could save and move from waste disposal budget to other categories. one thing we are looking into are filter vials.
they are safer to use. require less storage volume. require less bench volume to work with (that is the most expensive thing in a lab)
they make less waste. a lot less waste.
for sample that are 100% water based and mostly water based, you can make the dilution steps directly into the filter vial with a pipettor. for the applications where it is possible it safes a bundle of time. less usage of volumetric flasks, less solvents, less pipette usage, less dishwasher waste. less sample liquid waste.

because it was effecting an indirect lab budget, it was very hard to convince management of the idea. it actually took a safety accident of a filter exploding in someone's face to get things moving.

[vahur]

Dear Vahur,

When you state that changing the column is not an option does that relate to;

1) That specific brand,
2) That type of stationary phase,
3) Specific column dimensions,
4) Is it a pharmacopoeial method or fully validated method?

Thanks,
Scott

It´s fully validated and accreditated and buying a new column is not an option. Does anyone know if changing accreditated method before the next accreditation is legal or you just have to wait for next one and use new parameters then?

[tom jupille]

If it's a regulatory question, what about getting a longer column? (you didn't say, but if it was a 150mm you could go to 250mm. The USP and EP allow column length to be adjusted +/- 70%, and the US FDA has a similar limit for their internal use (see attachment A to document ORA-LAB.5.4.5:
http://www.fda.gov/downloads/ScienceRes ... _content=3

So long as you meet system suitability, that +/- 70% is considered an "adjustment" and does not require revalidation.

[Scott F]

It really depends on the regulatory body whose jurisdiction you fall under. Some regulatory authorities permit small changes such as those detailed in the System Suitability section of the Chromatography Chapter (621) of the USP. I would actively recommend looking into the exact validation report and if necessary speaking with appropriate regulatory / accreditation contact you have for you laboratory - you will be amazed as some are very helpful........

Please follow the below links to some really useful articles detailing changes that can be made but please do bear in mind these will potentially not only have to approved bu the external regulatory / accreditation body, but you own quality / regulatory depts., which from my experience can be the most conservative!!


http://www.pharmtech.com/pharmtech/data ... rticle.pdf

http://www.layloff.net/fda/1998SystemSustainabilty.pdf

http://www.waters.com/webassets/cms/lib ... rticle.pdf

Please post back with how you're getting on,

Scott

[vahur]

Thanks for all the links I´ll work them through.

Had time today to test different temperatures. If I got it right the resolution is best at 30C and the separation of peaks is best at 20C. I uploaded the results to googledocs. The sensitivity appears to be lower at lower temperatures. didn´t have time to test LoD but it could become an important factor.

https://docs.google.com/spreadsheet/ccc ... E5sR0NWZ3c

Edit:
One more thing. The separation is worse with real samples which probably comes from not using a buffer solution instead of water. Don´t know what´s the reasoning for using water.

[Scott F]

Not unsurprising that whilst selectivity increases at lower temperature, LOQ decreases and chromatographic efficiency reduces to such an extent that resolution also reduces.

Have you had chance to look through the previously posted docs yet - do you have any scope for making changes to you mobile phase / column?

Regarding your second point - are you referring to using water instead of a buffer solution as the sample solvent or mobile phase? You question seems to indicate you are referring to sample solvent but I seem to remember your weak mobile phase component was pure water. If it is related to the mobile phase, please remember the purpose of a buffer. It is present to maintain the pH at a specific value, close to the buffers pKa, in order that your analyte(s) are present in a single ionic form. If your analytes are non-ionisable or their pKa values are more than 2 units away from the water : organic solvent mixture, buffers are not always required. Having said that it is good practice to employ a buffer, usually at the lower end of the pH scale in order to control the ionisation state of the resuduals silanols in your column when analysing not just acidic but basic compound too.

Scott

[vahur]

Not unsurprising that whilst selectivity increases at lower temperature, LOQ decreases and chromatographic efficiency reduces to such an extent that resolution also reduces.

Have you had chance to look through the previously posted docs yet - do you have any scope for making changes to you mobile phase / column?

Regarding your second point - are you referring to using water instead of a buffer solution as the sample solvent or mobile phase? You question seems to indicate you are referring to sample solvent but I seem to remember your weak mobile phase component was pure water. If it is related to the mobile phase, please remember the purpose of a buffer. It is present to maintain the pH at a specific value, close to the buffers pKa, in order that your analyte(s) are present in a single ionic form. If your analytes are non-ionisable or their pKa values are more than 2 units away from the water : organic solvent mixture, buffers are not always required. Having said that it is good practice to employ a buffer, usually at the lower end of the pH scale in order to control the ionisation state of the resuduals silanols in your column when analysing not just acidic but basic compound too.

Scott

Yes, I read throug those and they were very informative. Thanks alot. Now I´ll have to find out who regulates my accredutation and what are the exact rules.
I was referring to mobile phase. Maybe I´m getting it wrong but the matrixes I´m analysing are pretty different(alcoholic beverages, juices, foods)which means they can have different pH-s. So When I inject my sample the sample pH can affect the mobile phase pH when I´m not using a buffer and make my retention times shift from sample to sample. As both of my analytes have an ionizable group they can be affected by the injection pH and move differently at the start of the column.
We are also using automated integration system, which uses retention times to identify peaks. If buffer was used I wouldn´t have to recalibrate the times so often.

[zheyin]

Not unsurprising that whilst selectivity increases at lower temperature, LOQ decreases and chromatographic efficiency reduces to such an extent that resolution also reduces.

Have you had chance to look through the previously posted docs yet - do you have any scope for making changes to you mobile phase / column?

Regarding your second point - are you referring to using water instead of a buffer solution as the sample solvent or mobile phase? You question seems to indicate you are referring to sample solvent but I seem to remember your weak mobile phase component was pure water. If it is related to the mobile phase, please remember the purpose of a buffer. It is present to maintain the pH at a specific value, close to the buffers pKa, in order that your analyte(s) are present in a single ionic form. If your analytes are non-ionisable or their pKa values are more than 2 units away from the water : organic solvent mixture, buffers are not always required. Having said that it is good practice to employ a buffer, usually at the lower end of the pH scale in order to control the ionisation state of the resuduals silanols in your column when analysing not just acidic but basic compound too.

Scott

Yes, I read throug those and they were very informative. Thanks alot. Now I´ll have to find out who regulates my accredutation and what are the exact rules.
I was referring to mobile phase. Maybe I´m getting it wrong but the matrixes I´m analysing are pretty different(alcoholic beverages, juices, foods)which means they can have different pH-s. So When I inject my sample the sample pH can affect the mobile phase pH when I´m not using a buffer and make my retention times shift from sample to sample. As both of my analytes have an ionizable group they can be affected by the injection pH and move differently at the start of the column.
We are also using automated integration system, which uses retention times to identify peaks. If buffer was used I wouldn´t have to recalibrate the times so often.

I don't see any ionizable group on D2 and D3.
pH of your mobile phase won't affect.