Chromatography Forum

Hi everyone,

Please I want your help on a certain problem

I want a method for analysis of Metformin HCl and Vildagliptin by HPLC

The problem is that Metformin retention time is very low.....so I use ion pair or/and TEA to make it more retained but this results in tailing of the peak (very broad)

I use 2 wavelength: 200 nm for Vildagliptin (it has very low absorbance)
& 260 nm for Metformin

The formula contains 1000mg Metfromin and 50mg Vildagliptin

Thanks for your support.

I did a quick Google search on "metformin" +"HPLC" and turned up at least a half-dozen methods on the first page. Retention on C18 column does not seem to be a problem using ACN/phosphate buffer systems (no ion-pair reagents needed). That suggests that you may have a dead (or inappropriate) column.

1st: Thanks for your help.

2nd: I tried most of the methods which I found on Google, also I tried different C18 columns from different suppliers.....Metformin is not retained unless I use ion pair reagent

Even when not using ion pair the Metformin peak has a large tail.

amirnimo,
I cut the application from Agilent and put it here.

and

Both of the application use reverse phase column as tom jupille suggested you but use MS.

and here for my condition is,

For Metformin present at 1.64 min and it seem be symmetry.

Best Wishes
Jetjamnong

Given the very low logP of metformin (-1.5, logD at pH 6, -3?) I am surprised it is retained in RPLC. Perhaps the actual retention mechansim on RPLC sorbents is ion exchange with residual silanols.

HILIC is likely a more appropriate and perhaps more robust separation mode for this analyte.

Here is robust separation with mixed-mode. Retention comes from weak reversed-phase and strong cation-exchange. Other buffers can be used with the same results:
http://www.sielc.com/Compound-Metformin.html

Metform can be easily analyzed on Acclaim Trinity P1 column (see Figure 8 from http://www.dionex.com/en-us/webdocs/707 ... 239-02.pdf). This column is designed for API and counterions and other applications that involve charged molecules. The chromatographic condition is compatible with UV, CAD, ELSD and MS, and requires NO ion pair agent in the mobile phase. The retnetion time can be adjusted to be longer or shorter, by changing the buffer concentration or organic solvent in the mobile phase.

Hi,
I also meet this problem when I used some methods to analysis MET_VIL in pH 4.5 but tR of Met as same as tR of pH 4.5. So I used ion pair to make long tR of MET but pic VIL no appear.
Please help me to resolve this.
Thank for your helping!

Hi DIEGC40,

What are the separation conditions that you are trying to use, column phase, column dimensions, elution program, eluent flow rate, temperature, injection volume, detection wavelength?

Hi DIEGC40,

Just checking to see if you're still out there and interested in separating Metformin HCl and Vildagliptin by HPLC.

A HILIC method was reported:

https://www.sciencedirect.com/science/a ... 3214004243

Journal of Chromatography B, Volume 965, 15 August 2014, Pages 133-141, "Simultaneous determination of metformin and vildagliptin in human plasma by a HILIC–MS/MS method."

Also, UV-Vis methods were reported:

https://www.sciencedirect.com/science/a ... 2514000766

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Volume 125, 5 May 2014, Pages 175-182, "Validation of different spectrophotometric methods for determination of vildagliptin and metformin in binary mixture."

Hi Mattmullaney,
Thank you for your information
I used C8 column, mobile phase NaH2PO4: MeOH 85:15, V = 1,2 ml/min
But tR MET is as same as tR pic pH 4,5. So It is difficult to do.
Method HILIC_LC_MS: my university don't use
Method UV is not able to eluent 2 pic: Met and Vil
Could you share me the method which using HPLC?
Thanks.

Hi again, DIEGC40,

I think I understand, you cannot separate successfully metformin and vildagliptin via the use of a C8 column without the Metformin peak eluting at the void time. This makes sense as the metformin is quite polar, so an ion-pairing agent is needed or perhaps a different stationary phase altogether.

The HILIC (HILIC is an HPLC separation mode) phase should work well, or perhaps a SCX phase, if the idea is to perform the separation without using an ion-pairing agent. Please note, MS detection is not mandatory for use of a HILIC separation, UV detection will work okay, I'd think, though these substances don't have much in the way of UV chromophores. It seems likely that low UV (< 220 nm) would be amenable to detect both metformin and vildagliptin.

Like Tom noted above, there are some metformin and vildagliptin methods just when you search using Google...these call for UV detection between 215 - 260 nm...checking out the UV spectra of metformin and vildagliptin would be a good start, I think. None of the methods I found easily had ion-pairing agents...and none had metformin eluting anywhere outside of the column void.

Are you prohibited from using ion-pairing, or to use a phase other than C8?

Hi Mattmullaney
We are doing invitro test, at pH 1,2 and 6,8: our method is okay, but only pH 4.5 is not okay
So I still keep my idea which using ion-pairing. I will try again
Can you help me a method Which you used to do in the case?
Thank you very much!

Hi Mattmullaney
I used another stationary phase but it was not successfull
When I used ionpairing, tR Metformin is not at the void time.
Now, I am obstructive...

Hi Again,

Very annoying, lost my post somehow. Here are two ion-pairing methods for metformin:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246406/

https://www.ncbi.nlm.nih.gov/pubmed/12560065

I'm not happy about the pH control for either method but these could be a good starting point.