Biology Talk

palpandi · Guest

We are currently working on the optimization of production of statins.
Initially we have taken the natural statin (lovastatin) for the study.
Production is carried out in submerged fermentation with the fungus
Monascus purpureus MTCC 369. Analysis of lovastatin is performed in
HPLC (Shimadzu, Luna C18 column) with UV detector. We have our
standards in both open ring (â-Hydroxyacid) and closed ring lactone
forms, dissolved in acetonitrile. The mobile phase used is 60:40
acetonitrile and 0.1% orthophosphoric acid. The open ring form gets
eluted at 8th minute and the lactone form eluted at 11th minute.
Samples taken from the broth have been analyzed with assays given in
various literatures. Sample of a medium when injected, we got the
elution at the time of the lovastatin standard which gave a
confirmation of the compound produced. But when the same sample was
analyzed on the next day for the concordance we haven't got the same
area as before. Sometimes the standard solution (â-Hydroxyacid) is
getting eluted earlier at 6th minute rather than at 8th minute. The
problem is that we could not fix a particular method for the analysis
of the sample collected from the broth. We will be very grateful to
you if you could suggest any procedures that could be very effective
for us to analyze the samples. We have some other queries that we
would like to ask in this work are...
1. Will the standard elution time vary as when it is left unused for a
long time?
2. Why there is a change in the retention time of the same sample when
analyzed the next day?
3. What might be the reason for the variation in area of peaks
obtained for the samples when it is tested on the next day and how it
could be resolved?
4. How can we store the samples and standard solutions without any
deformities?

N10 · Guest

Hello Palpandi

Temperature variation of your system or parts of your system during
sequential instances of analysis might account for some of the phenomena
you mention.

A 2 minute variation in elution time is a very significant deviation which
suggests that polarity of your target had significantly changed. I would
try experimenting with an secondary internal standard to atleast confirm
relative rention times on subsequent days of analysis and also to look at
the ratios of relative peak areas. If you dont see a good correltion on
realtive rention time over time then your target molecule has almost
certainly changed.

Some other relevant factors.

Do you leave you pump running over night ?

How old is you column and pre column ( if you have one)

Do you de-gas or otherwise clean un your sample ?

Hope these observations and thoughts help.

Regards N10

"palpandi" <palpandi@gmail.com> wrote in message
news:41324459-dd06-411b-8dd3-4f82eaae50b6@c19g2000prf.googlegroups.com...
We are currently working on the optimization of production of statins.
Initially we have taken the natural statin (lovastatin) for the study.
Production is carried out in submerged fermentation with the fungus
Monascus purpureus MTCC 369. Analysis of lovastatin is performed in
HPLC (Shimadzu, Luna C18 column) with UV detector. We have our
standards in both open ring (â-Hydroxyacid) and closed ring lactone
forms, dissolved in acetonitrile. The mobile phase used is 60:40
acetonitrile and 0.1% orthophosphoric acid. The open ring form gets
eluted at 8th minute and the lactone form eluted at 11th minute.
Samples taken from the broth have been analyzed with assays given in
various literatures. Sample of a medium when injected, we got the
elution at the time of the lovastatin standard which gave a
confirmation of the compound produced. But when the same sample was
analyzed on the next day for the concordance we haven't got the same
area as before. Sometimes the standard solution (â-Hydroxyacid) is
getting eluted earlier at 6th minute rather than at 8th minute. The
problem is that we could not fix a particular method for the analysis
of the sample collected from the broth. We will be very grateful to
you if you could suggest any procedures that could be very effective
for us to analyze the samples. We have some other queries that we
would like to ask in this work are...
1. Will the standard elution time vary as when it is left unused for a
long time?
2. Why there is a change in the retention time of the same sample when
analyzed the next day?
3. What might be the reason for the variation in area of peaks
obtained for the samples when it is tested on the next day and how it
could be resolved?
4. How can we store the samples and standard solutions without any
deformities?