HPLC/chitosan/pectin

[Samoborac]

Hello,
I am having problem with HPLC analysis and I would highly appreciate some advice…
I am performing the dissolution studies of the drug from polymeric matrices obtained by the use of hydrophilic polymers, namely chitosan and amidated pectin. The dissolution media is simulated saliva (phosphate buffer pH 6.5 without enzymes).

The drug quantification was performed by HPLC on Purospher STAR RP-18 column (column size 125/4 mm, granule size 5 microm). The mobile phase is methanol:0.01 M phosphate buffer pH 6.8 in ratio 70:30, flow rate at 1.5 mL/min, detector at 235 nm (absorption maximum of the drug), column temperature 40C, injection volume 20 microL. I have successfully validated the method and I have obtained excellent results.

Than I have started the dissolution studies. And I have noticed that the pressure of the system is constantly increasing… During the validation of the method it was stabile, around 16.5 MPa, but when I started to analyze the samples containing the amidated pectin and chitosan, the pressure was risen up to 25 mPa… And this pressure increase is constant, not only during the injection of the sample. Is it possible that pectin is blocking the column? All samples have been filtrated prior to injection.
The solubility of chitosan in the dissolution media is low; therefore I think that he should not be the cause. I have tried everything, the back-flushing of the column with the water (pectine is soluble in water), the washing of the column according to the manufacturers advice (water-methanol-acetonitrile-methanol-water), have purged the tubes but the pressure is still high. Moreover, I have observed the tailing of the pick (at concentrations that are in the middle of the working range. For the same drug concentration, during method validation period, the peak was symmetrical)…
The mobile phase is stabile, during the 2 days after mixing the components I have not notice crystallization of the salts from the buffer. Addition of the simulated saliva to mobile phase does not change its appearance. At the end of each working day, I have washed the column by 30 ml of methanol/water (70:30) mixture followed by 30 mL of methanol/water (30:70) and finally by 30 mL methanol/water (70:30). The producer of the column has stated that 30 mL is the washing volume for this column… I think that by this procedure I should have removed buffer and remaining polymer by this washing.

Is it possible that pectin is messing up with the silica? Did anybody have similar problems? Any advices about performing the HPLC analysis in the presence of hydrophilic polymers? How, I have ordered the new column, but I have fears not to destroy it… What am I doing wrong?

[Bryan Evans]

Can you put a guard cartridge in front of the analytical column?
You may be able to extend the life of the analytical column.

Other options (though not sure how well they will work):
- increase % water in mobile phase
- increase column temperature
- go to 3um (and reduce inj. volume)

[orcicdejan]

Have you checked the solubility of your sample (the one ready for injection, drug @ simulated saliva) in the mobile phase? Filtering won't help if your sample precipitates after the injection. The guard column would be a workaround in this case, but you should make certain that no precipitation occurs.

[Bryan Evans]

Good call, I should've mentioned that. You can check solubility of your
sample / mobile phase in test tubes (not your LC). It's cheaper and faster.

[Noser222]

I've worked a lot with pectins as part of a formulation and I'd adivse you to use a gradient method starting out < 10% methanol. Hold at < 10% methanol for at least 2 column volumes and it should wash away.
As for recovering the column that you are using, backflushing with water, maybe even ammonium acetate at 100 mM, is probably all you can do. It could take a day or so; monitor your pressure to see if it returns to normal.

[Samoborac]

Hey everybody,

Thank you very much for the responses posted, they were of great help…


Yes, I have checked the miscibility of drug solution in simulated saliva with mobile phase and it was OK. It seems that pectin is the source of the problem, its solubility in the mobile phase seems to be limited…
I will try to increase the % of buffer in the mobile phase, but that will prolong the retention time (there is no auto-sampler)… And in the lab were I am now (as a PostDoc student) they do not use pre-column… I don’t think that this is a good approach, but I couldn’t do much… I will try to bring the column back to life by the washing procedure proposed by Noser 22
And the instrument does not have the option of the gradient mode… Could be sufficient if I backflush the column with 60 mL of the MeOH:water (10:90) at the end of each workday?

[orcicdejan]

Well, as for washing, i use simple but efficient procedure: wash until the pressure and the signal from detector are stable (you may repeat it with different mob. phase compositions).

[HW Mueller]

It looks like the filtration probably didn´t get rid of the polymers maybe you need an ultrafiltration, SPE, SEC, or liquid-liquid extraction step. I am not familiar with pectin, etc., but can immagine that these collect on the column. Even extremely low amounts of polymers can cause problems if they accumulate on the column.

[Samoborac]

The filtration of the samples would not solve the problem because the pectin is dissolved (not dispensed) in the simulated saliva, therefore it can’t be removed by filtration.
Regarding the washing procedure for the column, what do you recommend as a optimal flux rate… Now I have spoken with a dealer of the column and he has suggested 0.2 mL/min…. And really, when I was doing the washing at the high flux /1.5 mL/min) the base line was stable, now when I have reduced flux to 0.2 mL I see numerous intense peaks….

[Bryan Evans]

You can "counter" the increase in % aqueous with less retentive column
(e.g. C8, phenyl).

No promises though. Your chances of success (with limited sample pretreatment)
are greatly reduced without gradient elution capabilities.

[ElizCates]

Samoborac commented:

The filtration of the samples would not solve the problem because the pectin is dissolved (not dispensed) in the simulated saliva, therefore it can’t be removed by filtration.

Standard filtration might not do the trick, but membrane filtration probably would. Dialysis tubing or a comparable size-exclusion membrane filter would probably neatly separate the pectin out from the analytes of interest.

Good luck!

[Samoborac]

Thanks for the hints...

The most confusing for me is the fact that I have found several publications (i.e. Anrabi et al., Eur. J. Pharm. Sci. 10 (2000) 43) where authors have performed the dissolution studies of the pectin based formulation by direct injecting of the samples colected in simulated gastric and simulated intestinal fluid, without any preatreatment... They have only treated the samples colected in simulated cecal fluid, to precipitate the pectinolytic enzymes that they have added to the dissolution media. They did not use the gradient method... I have sent the query to the correspondin author, but I didn't get any responce.

Now I am thesting the behaviour of the polymer solution in the simulated saliva in different mobile phase... Perhaps the use of acetonitrile and higher amount of the water would do the trick...

[HW Mueller]

That you didn´t get an answer speaks for itself. You may be throwing good money after bad. My suggestion: Get rid of any macromolecules, including proteins from body fluids, before doing a normal chromatography. Even these internal RP-surface exclusion columns (which are supposed to get rid of proteins, .....) often produce flow problems.

[Noser222]

Not that SEC/ultrafiltration isn't the best way to go for preserving the column....

Pectin can be washed away. We worked with the worst pectin imaginable for reverse phase and a wash step at the beginning with < 5% acetonitrile or < 10% methanol with unbuffered ammonium acetate was enough for us to wash it away and then transition to the mobile phase we needed for the analytes.

[Noser222]

ah, glanced over and missed the part about no gradient capability...