Lubiprostone HPLC method

[wkhuang]

I'm developing an HPLC method for Lubiprostone assay and impurity method in a liquid dosage form.

My sample concentration is very low and has lots of other fatty acids in the sample solution. I am not able to get UV response. Does anyone offer advice? Thanks.

[krickos]

Hi

I do not have a direct solution for your problem but might get you thinking abit.

First, the molecule has not many chromofores (2 carbonyls, no aromatic ring structure etc) so UV detection is obviously harder. You may want to look into another detectors (MS, ECD,RI, ELDS).

As for sample preparation it is hard without details, but can give you a not uncommon example including removal of FA droplets, assuming you talk about some capsule with the acive ingredient in FAs.

Dissolve a suiteble number of capsules in diluted not too strong acid (example phosphoric) or weak base if more suiteble to obtain desired concentration.
Dilute with mobile phase.
Filter (suiteble syringe filter or perhaps SPE) sample solution carefully to avoid breakthrough of FA droplets.

Given that the product/drug substance is relative new if I saw right, the amount of open information may be limited.

[carls]

This compound is relatively hydrophobic even when the acid group is deprotonated (pKa ~4.5). However, it is less hydrophobic than your fatty acids especially at pH >6.5. Also this compound is relatively non volatile so CAD (better sensitivity than ELSD) could be used instead of UV, however, its hard to say which (CAD/UV) will give better sensitivity.

You could use RP-SPE (C18 silica or non-derivatized polymeric) to retain the fatty acids while allowing your active to pass thru by finding the right organic concentration in the load solvent.

You could try UV at 200nm if using ACN and H3PO4. I suggest you look at the UV spectrum in ACN/H2O to find the best low UV wavelength.

[carls]

In the above post I assumed you wanted to removed the MCT's.

However, if yor objective is to conentrate the active then anion exchange SPE could be used. For example, dissolve the liquid formulation at the highest concentration that is miscible with 50/50 ACN/5mM pH 7.5 phosphate buffer. Condition SPE with methanol followed by buffer. Load sample (volume depends on sorbent mass/volume of SPE device). Wash with 100% MeOH. Elute with minumum volume 0.1N HCl in ACN. Evaporate to dryness. Reconsitute to minimum volume (200uL?) with solvent that redissolves analyte of interest and is appropriate for the HPLC method.

Hope this helps

[wkhuang]

Carls/Krickos, Thanks a lot for your advice. I tried CAD and it seemed to work. But I still have a long way to go since my sample concentration is just too low. It also turned out that the inactive ingredients are not free fatty acids but medium chain triglycerides.

[krickos]

. It also turned out that the inactive ingredients are not free fatty acids but medium chain triglycerides.

Actually the example given by me above with the weak acid comes from some soft gelatin capsule with medium chain triglycerides, memory is vague though as we stopped analysis of that one some time ago.

See it like this, if you do a good job on the final product testing the QC staff will likely complain more about the time spent on full specification testing of the medium chain triglycerides according to Ph Eur.

[GaneshRAJ]

I'm developing an HPLC method for Lubiprostone assay and impurity method in a liquid dosage form.

My sample concentration is very low and has lots of other fatty acids in the sample solution. I am not able to get UV response. Does anyone offer advice? Thanks.

Hi wkhuang,
I had gone through your concern with respect to analytical method for assay and Related substance. I also finding the same problem so need to know whether it has resolved from your end regarding analytical method and if yes can u share some thoughts that will help me to work on as the sample concentration is very low and detection at low level with UV found to be more difficult.

[Murshad]

I'm developing an HPLC method for Lubiprostone assay and impurity method in a liquid dosage form.

My sample concentration is very low and has lots of other fatty acids in the sample solution. I am not able to get UV response. Does anyone offer advice? Thanks.

Hi wkhuang,
I had gone through your concern with respect to analytical method for assay and Related substance. I also finding the same problem so need to know whether it has resolved from your end regarding analytical method and if yes can u share some thoughts that will help me to work on as the sample concentration is very low and detection at low level with UV found to be more difficult.

Hi folks
Any luck on your side. I really wanted to know how far you guys have gone so far. I came across during my casual search some method using 205nm and acetonitrile as mobile phase. With acetonitrile and this UV would not be a smooth ride for a bench chemist.
Did you guys ever thought of derivatizating the active or using LC-MS way
Regards

[Vlad Orlovsky]

If triglycerides are giving you a problem you can switch to anion-exchange mixed mode column (Coresep SB, Heritage MA) and use high organic/low ion concentration to elute your triglycerides first and then elute your target which will retain by combination of weak RP (since you have a lot of ACN) and medium strength anion-exchange.

Contact me if you have any questions