Chromatography Forum

I was trying to work on the USP Clarithromycin method for RS (USP28, Supplement2). I am facing a very peculiar problem I have used Hichrome KR100 KromasiL C18, 100 x 4.6mm, 3.5µ. It gives excellent RESOLUTION as required in the Identification solution, required peak tailing. The problem is that Column goes bad only after 20-25 injections. I have consumed 4-5 new columns and observed the same problem.

HAS somebody faced a similar problem, and how to improve the column life.

PLEASE SUGGEST THE WAY OUT.

In other aspects USP method is quite good.


Thanks in advance.

What are the problems observed with the column: peak tailing, efficiency loss, decrease in resolution; high pressure? Have you tried different manufacturers/batch column.

Resolution between the closest eluting peaks goes bad. The tailing increases and also the peak shape deteriorates drastically.

I have tried Hichrome KromasiL 5 columns and Flexit kromasiL 2 columns . All the columns go bad after about 20-30injections and interestingly all show excellent resolution and peak shape at the start.

Am I missing something or some special care has to be taken.

Pawan- You have done Clari - so many times. Is thare any specific way to clean the column.

Thanks

Shashi Kant Tiwari.

Resolution between the closest eluting peaks goes bad. The tailing increases and also the peak shape deteriorates drastically.

I have tried Hichrome KromasiL 5 columns and Flexit kromasiL 2 columns . All the columns go bad after about 20-30injections and interestingly all show excellent resolution and peak shape at the start.

Am I missing something or some special care has to be taken.

Pawan- You have done Clari - so many times. Is thare any specific way to clean the column.

Thanks

If ur analyte clarithromycin is highly hydrophobic try flushing out with a strong solvent.


have proper time gaps between two consecutive injections...prevents column loading.

did u try injecting the concentration lower than what u had been injecting?


regards

amaryl

I have never used USP Supplement method.
But is there any specific purpose of passing mobile phase thro C18 prior to analysis.
You can contact me at pkratra1@yahoo.com

I don't have access to a copy of the method, but the last comment about passing the mobile phase through a C18 column first piqued my interest. What is the mobile phase that's causing the problem?

Mobile phase— Prepare a mixture of methanol and 0.067 M monobasic potassium phosphate (650:350), adjust with phosphoric acid to a pH of 4.0, filter through a filter having a porosity of 0.5 µm or finer, and degas. Make adjustments if necessary (see System Suitability under Chromatography <621>).

Your symptoms are consistent with development of a column headspace; I was wondering if your mobile phase had a high pH Aside from the obvious poor practice (phosphate is not a good buffer at pH 4, and pH adjustment after adding organic adds uncertainty), I don't see anything there that would obviously shorten column lifetime.

I know that the other methods can be developed. The problem is that this is USP method ( and also the EP Method) for Clarithromycin. In any case we have to follow this. As I mentioned earlier the method works well on the expense of column life.

Has anybody similar experience.

Shashi Kant

We still don´t know whether there is a pressure change concurrently with peak deterioration, nor whether the column is restored by running MeOH, etc. through. What´s the matrix you are injecting?

Can we do something about these straight-jackets of authorities? We seem to see continuously more of these. Hard to believe that a good intention was behind this (stop fraudulent analytics....).
(The road to hell is paved with good intentions?)

Gee....

The method specifies a pH of 4.0 with phosphate? No wonder the method is irreproducible. At pH 4, phosphate has no buffering capacity.

I suggest you develop your own method based on sound principles of basic chemistry.

FOLLOWING IS THE USP METHOD ATTACHED - PART ------------------------------------------------------
Solution A— Prepare a solution containing 4.76 g of monobasic potassium phosphate per L. Adjust with dilute phosphoric acid (l in 10) or potassium hydroxide (45% w/v) to a pH of 4.4. Pass this solution through a C18 filtration kit.
Solution B— Use acetonitrile.
Mobile phase— Use variable mixtures of Solution A and Solution B as directed under Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621 ).
Diluting solution— Prepare a mixture of acetonitrile and water (50:50).
Standard solution A— Transfer about 75 mg of USP Clarithromycin RS, accurately weighed, to a 50-mL volumetric flask, and dissolve in 25 mL of acetonitrile. Dilute with water to volume, and mix.
Standard solution B— Transfer 5.0 mL of Standard solution A to a 100-mL volumetric flask, dilute with Diluting solution to volume, and mix.
Standard solution C— Transfer 1.0 mL of Standard solution B to a 10-mL volumetric flask, dilute with Diluting solution to volume, and mix. This solution contains about 0.0075 mg of USP Clarithromycin RS per mL.
Standard solution D— Transfer about 15 mg of USP Clarithromycin Identity RS, accurately weighed, to a 10-mL volumetric flask, dissolve in 5.0 mL of acetonitrile, dilute with water to volume, and mix.
Test solution— Transfer about 75 mg of Clarithromycin, accurately weighed, to a 50-mL volumetric flask, dissolve in 25 mL of acetonitrile, dilute with water to volume, and mix.
Chromatographic system (see Chromatography 621 )— The liquid chromatograph is equipped with a 205-nm detector and a 4.6-mm × 10-cm column that contains packing L1 and is maintained at a constant temperature of about 40. The flow rate is about 1.1 mL per minute. The chromatograph is programmed as follows.
Time
(minutes) Solution A
(%) Solution B
(%) Elution
0®32 75®40 25®60 linear gradient
32®34 40 60 isocratic
34®36 40®75 60®25 linear gradient
36®42 75 25 isocratic

Relative retention times with reference to clarithromycin (retention time = about 11 minutes) include the following: impurity I = about 0.38; impurity C = about 0.89; impurity F = about 1.33; impurity A = about 0.42; impurity D = about 0.96; impurity P = about 1.35; impurity J = about 0.63; impurity N = about 1.15; impurity K = about 1.59; impurity L = about 0.74; impurity E = about 1.27; impurity G = about 1.72; impurity B = about 0.79; impurity 0 = about 1.38; impurity H = about 1.82; and impurity M = about 0.81.
System suitability— Chromatograph Standard solution B, and record the responses as directed for Procedure: the tailing factor for the main clarithromycin peak is not more than 1.7. Chromatograph Standard solution D, and record the responses as directed for Procedure: the peak-to-valley ratio ( H P / H V ) of impurity D and clarithromycin is not less than 3.0, where H P is the height above the baseline of the peak due to impurity D; and H V is the height above the baseline of the lowest point of the curve separating this peak from the peak due to clarithromycin.
Procedure— Separately inject equal volumes (about 10 µL) of the Diluting solution, Standard solution B, Standard solution D, Standard solution C, and the Test solution into the chromatograph, record the chromatograms, and measure the peak area responses. Calculate the percentage of each impurity in the Clarithromycin taken by the formula:
50( C C / W)( r i F / r C )P,
in which C C is the concentration, in mg per mL, of USP Clarithromycin .

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As told earlier This method is currently official in USP and EP. Hence in all cases we have to follow this as this method can separate many impurities which will be diificult to synthesize and required for development of an IH method for these.
If we are using USP method, RRT Mentioned here can solve the purpose of identification for such impurities.

Rgds
Shashi Kant Tiwari

I still think that the major problem is the lack of proper pH control. However, the method is also carried out at 40 degrees C. Under these circumstances, changes in silanol population may happen more frequently than at room temperature.

Have you ever tried to run the method at room temperature?

The Kromasil C18 is quite a decent packing. However, if I recall correctly, it is based on a monofunctionally bonded C18. Other C18's could provide a higher stability, if at all there is something to the stability of the column itself. Try to find a trifunctionally bonded C18! However, it is not clear if the correct selectivity would be preserved if you do that.

I believe that the C18 filtration is to remove UV-absorbing impurities in the buffer. At 205 nm, you need all the help you can get.

Our company and I have an experience on that API.
I had also faced that kind of problem. I resolved the problem by reducing the injection volume about half-fold.
So, I suggest that you reduce the injection volume as half and increase the sensitivity twice.