Strange peak splitting

[syx]

Syx, what do they call the cis and trans isomers. At what point in the molecule is the cis-trans transformation? I am used to cis-trans around a double bond, but I can't see it here?

The cis-trans position of methyl and H in cyclohexyl ring (upper right). Cis-isomer is process impurity, not a degradate.

... if you are referring that article over isomer determination in crude samples of glimepiride, the one I send to you, thats RP HPLC.

Here are some articles related to Glimepiride isomers and the used condition for separation:
Song Y, et al. J.Sep.Sci. 2003, 26, 1595-1597
Column: Dikmonsil C18 5um 250 x 4.6 mm
Mobile phase: Methanol:acetonitrile:HOAc-NH4Ac buffer (1.5M , pH 4.5) = 1.1:1.3:1.0
Flow rate: 1.0 mL/min
Injection volume: 10 uL (sample concentration 100 ppm)
Result: Rt of trans-isomer = 23.018 min; cis-isomer = 24.885 min

Jun W, et al. Chinese Journal of Spectroscopy Laboratory 2004, 21(1) 150-152
Column: Zorbax SB-C18 (250 x 4.6 mm, 5um)
Mobile phase: Methanol: HOAc-NH4Ac buffer (0.05M, pH 3. = 68:32
Flow rate: 1.0 mL/min
Temperature: 20 C
Result: Rt of trans-isomer = 22.4 min; cis-isomer = 23.7 min

Khan MA, et al. J.Pharm.Biomed.Anal. 39(2005) 928-943
Column: Phenomenex Luna C8(2) 5 um, 250 x 4.6 mm
Mobile phase: Phosphate buffer (pH 7.0):acetonitrile:tetrahydrofuran = 73:18:9
Flow rate: 1.0 mL/min
Temperature: 35 C
Result: Rt of trans-isomer = 43.275 min; cis-isomer = 41.383 min

USP29–NF24 Page 1001
Column: 3-mm × 15-cm column containing 5-µm packing L20
Mobile phase: Transfer 100 mL of isopropyl alcohol into a 1-L volumetric flask, add 1 mL of glacial acetic acid, dilute with hexane to volume, filter, and degas.
Flow rate: 0.5 mL/min. [ NOTE— The analyses could also be performed with 4.6-mm × 15-cm, 4.6-mm × 25-cm, 4-mm ×12.5-cm, or 4-mm × 25-cm columns containing packing L20. It is recommended that the flow rate be adjusted to about 1.1 mL/min for a 4.6-mm column and to about 0.8 mL/min for a 4.0-mm column.]
The relative retention times are 1.0 for glimepiride and not more than 0.9 for the cis-isomer, and the signal-to-noise ratio of the cis-isomer peak is not less than 15.

See? Need a lot of time to separate the isomers.

the compound whose chromatograms i posted in the very begining was Amlodipine Besylate but we have observed this with Ramipril.

Btw, have you any reference about substances (excipients, especially in tablets) those could adsorb amlodipine? I have difficulty to get good recovery. I always got below 98.0% though I avoid filtration and use many types of solvent.

[HW Mueller]

Well, I just mentioned temp. as one example, solvent variations, pH changes, diffs in metal or other ion trace content, dust particles, light ......... lots of things can catalyze or effect such reactions. I tried to be polite by saying something is fishy, actually I should have mentioned right away: If your stuff is stable during chromatography, as for instance, the good shape of Oliver´s peaks show, then you are handling the sample wrong (outside of the chromatography). (Actually I am still being polite, as students we used to call these bum problems).
(This pertains to the five membered ring)

[HW Mueller]

Ups, I actually had the "good shape" peaks of amaryl on my mind, above.

[amaryl]

Well, I just mentioned temp. as one example, solvent variations, pH changes, diffs in metal or other ion trace content, dust particles, light ......... lots of things can catalyze or effect such reactions. I tried to be polite by saying something is fishy, actually I should have mentioned right away: If your stuff is stable during chromatography, as for instance, the good shape of Oliver´s peaks show, then you are handling the sample wrong (outside of the chromatography). (Actually I am still being polite, as students we used to call these bum problems).
(This pertains to the five membered ring)

Handling the sample wrong (outside of the chromatography) means? what bum problems

Thanks and Regards,

Amaryl.

[amaryl]

Relatively recently published paper on analysis of this molecule. A quick skim reveals that their chromatography seems OK, but their choice of buffer for the pH they used is a bit odd I think (phosphate for pH 3.5?).

Still, here it is:

http://makeashorterlink.com/?C5552268C

Sir, you will find many such papers with loop holes ...pH out of buffer range... molarity of buffer too much. Fake papers !

Amaryl.

[HW Mueller]

amryl, not going into theories, on practical terms it is likely that your problem will go away like Oliver´s. If you observe carefully what you are doing you might even find out what went wrong, and something did indeed go wrong.

[amaryl]

amryl, not going into theories, on practical terms it is likely that your problem will go away like Oliver´s. If you observe carefully what you are doing you might even find out what went wrong, and something did indeed go wrong.

What all i remember is that I tried to observe the effect of pH changes one day and worked at pH 4 and 6. pH 6 was my last run that day. After that my every sample standard, even tablets, and degraded samples started exhibiting this ghost peak. The peak was small when i worked with HPLC-UV and at lower concentration of glime (below 1ug/ml) it disappeared.

Later when i worked on HPLC-PDA I got this result which i have pasted here of standard. The ghost peak well resolved from glime peak...where as in HPLC-UV it was close to glimepiride peak. Tailing of glime peak and emergence of ghost peak. Here the peak 1 was glime and peak 2 was ghost and vice versa with HPLC PDA. Peak 1 was ghost n peak 2 was glime.


I checked everything right from cleaning of flask, proper washing of column (even tried reverse flushing- column regeneration) changed solvent bottles ...even tried another standard, fresh tablets, all were in vain.

Regards,

Amaryl.

[HW Mueller]

Different columns and/or mobile phases on the different detectors? Detectors don´t do chromatography.

[amaryl]

Different columns and/or mobile phases on the different detectors? Detectors don´t do chromatography.

A shift of detector or other instrument was due to some other practical reason not due to ghost peak.

Well the different instrument resolved the two peaks nicely and it no more looked a ghost peak. There was no change in column and mobile phase conditions.

Regards,

Amaryl.

[HW Mueller]

????, but as predicted.

[syx]

... After that my every sample standard, even tablets, and degraded samples started exhibiting this ghost peak. The peak was small when i worked with HPLC-UV and at lower concentration of glime (below 1ug/ml) it disappeared.

Later when i worked on HPLC-PDA I got this result which i have pasted here of standard. The ghost peak well resolved from glime peak...where as in HPLC-UV it was close to glimepiride peak. Tailing of glime peak and emergence of ghost peak. Here the peak 1 was glime and peak 2 was ghost and vice versa with HPLC PDA. Peak 1 was ghost n peak 2 was glime.

... There was no change in column and mobile phase conditions.

The concentration of ghost is related to the main substance. It may be an impurity of the glimepiride or contaminant from dirty sample containers or spatulas.

I think your working concentration in this assay is too small. It means your method needs extra dilution steps that require more solvent consumption and give higher uncertainty. One must decide ecological and economical side of an analysis procedure.

Dissimilar result may be due to the different LC system and different batch of mobile phase.