Ketoconazole [May 3, 2004]
Posted: Wed Aug 25, 2004 4:38 am
By Anonymous on Monday, May 3, 2004 - 10:38 am:
How can I get a good peak shape for ketoconazole???... I've tested many different buffers, ph's and no success...
my mobile phase is ammonium acetate 10 mm + 1 mL of diethilamine / methanol
22:78
Column: C 18 250x4,6 X Terra
Is there any other better method you could suggest??
thanks
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Monday, May 3, 2004 - 03:29 pm:
We have run this compound without difficulty at alkaline pH on XTerra. What pH are you using? Alternatively, you can look at this application on Symmetry: http://www.waters.com/pdfs/Symmetry94.pdf
-------------------------------------------------------------------------------------------------------
By Carlos Teixeira on Tuesday, May 4, 2004 - 10:05 am:
Hello Anon-10:38;
X-Terra is a good (and roboust hplc-column), but, maybe you change Methanol by Acetonitrile? Wich Acn your chromatographic run will decrese (minor RTs). I beleave than with Acn your Ketaconazol peak will be better.
Good elutions!!
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 01:22 pm:
The problem is that i analyse creams and shampoo with no extra preparation, i just dilute and that's all..... but after a day of work i wash the column with water/meoh/acn/water and acn 30:70 for 2 hours.....
My x terra is new.... so that's why i'm surprising with the shape of the peak... I forgot to mention that when i chose this methodology, the peak was ok.....
the ph is 10
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Tuesday, May 4, 2004 - 04:34 pm:
I would think then that the problem is not that the column does not perform, but that something is accumulating on the column that your wash does not remove. Your wash is fundamentally OK, but maybe some assistance with the addition of acetic acid to your washes might help. I have to admit though, that this is a rather wild guess at what could be the problem.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, May 5, 2004 - 07:28 am:
i'll try this one (acetic acid)
thanks!
-------------------------------------------------------------------------------------------------------
By syx_gf on Thursday, May 6, 2004 - 06:13 pm:
These days I work with ketoconazole shampoo. I use diisopropylamine (2 ml/1 L MeOH) - ammonium acetate (1 g/200 ml water)(70 : 30). By using ZE (Agilent) column I have a good peak shape. The problem is there are three peaks from our formula that elute too close to the ketoconazole's.
I think acid is not a good choice, it will be tailing. Ketoconazole shows good shape in basic mobile phase (pKa: 6.51 and 2.94).
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, May 7, 2004 - 08:19 am:
hi syx... I'm going to use acetic acid just to wash the column because of the tailing that my tests are presenting.....
but thanks a lot for your help, especially for an option for another column...
And by the way... what's the pH of your mobile using diisopropylamine? mine is about 10.
-------------------------------------------------------------------------------------------------------
By syx_gf on Sunday, May 9, 2004 - 06:12 pm:
pH of this mobile phase is higher than 9. The best pH for ketoconazole based on its pKa is above 8.51 (pKa+2). In this pH, we can not use "general" column, but a basic resistance column. I think X-Terra is OK for this condition.
-------------------------------------------------------------------------------------------------------
By lime on Monday, May 10, 2004 - 06:54 am:
Column : C18, 125 x 4.0 mm, 5 µm
Wavelength : 240 nm
Flow : 1.5
Inj. Vol. : 10 µl
Column Oven : 45 ºC
Mobile phase : 6 g ammonium acetate in 380 ml water.Add 320 ml methanol and 300 ml acetonitrile
try it. Its been validated for ketoconazole supp.
good luck.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 11, 2004 - 07:44 am:
Hi lime...
i'll try this one...
Thanks a lot!!!
-------------------------------------------------------------------------------------------------------
By syx_gf on Tuesday, May 11, 2004 - 05:37 pm:
Finally, I decided to use diisopropylamine (2 ml/1 L MeOH) - ammonium acetate (1 g/200 ml water)(65 : 35)with gradient to higher organic conc after ketoconazole is eluted.
Column ZE (Agilent) C18, 5 um, 150 x 4.0 m
Flow 1.0
Temp 40
Work conc 40 ppm
Inj vol 20 uL
Rt Ketoconazole: 16.96', with Tf 1.18 and good resolution with our formula.
Lime, do you never have problem with your high conc buffer?
-------------------------------------------------------------------------------------------------------
By Lime on Thursday, May 13, 2004 - 07:13 am:
Hi !
Frankly, i have little problems with the column and i couldn't find out if it's about the mobile phase conc. or because we are working with supp. drug form.So, you might be right.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Sunday, May 16, 2004 - 08:39 am:
Lime: what is the pH of your ammonium acetate solution?
-------------------------------------------------------------------------------------------------------
By syx_gf on Tuesday, May 25, 2004 - 05:29 pm:
I think I should use SPE to eliminate other ingredients than ketoconazole. 45' total of my method is too long...
-------------------------------------------------------------------------------------------------------
By A.Mouse on Tuesday, May 25, 2004 - 06:13 pm:
What is your column length? Particle size? Why does it take so long?
-------------------------------------------------------------------------------------------------------
By syx_gf on Wednesday, May 26, 2004 - 12:40 am:
As I wrote on May 11. Sorry, Rt ketoconazole is 12.96, not 16.96. After 15' I use gradient to elute excipients behind the ketoconazole. (without gradient, they will elute until 90'!)
So, 30' behind is gradient section and re-equilibration.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Wednesday, May 26, 2004 - 02:52 pm:
Then why don't you do a step gradient to the final conditions just after ketoconazole elutes? If you do not care about information in the remainder of the chromatogram, no reason to try for high resolution in a 30 minute gradient.
-------------------------------------------------------------------------------------------------------
By syx_gf on Wednesday, May 26, 2004 - 05:26 pm:
I stop the chromatogram after ketoconazole is
eluted. Then I do the gradient for 15 minutes, and reequilibration for 15 minutes as the formula: ((10 x Vm)+Vd)/F.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Saturday, May 29, 2004 - 10:43 am:
There are several things that you can do to speed up the method. After your last peak elutes, you can use a step gradient to elute the junk. The step gradient can be done at high flow. Since you are using a 15 cm 5 micron column, this should not be a problem. Also the reequilibration can be done at high flow. Also, 5 column volumes may be sufficient for reequilibration.
Also, you may be able to run your method at higher flow rate. Of course, you need to check, if the resolution of all peaks is still fine.
If there is not a problem at all, other things can be done as well. We have converted many lengthy methods to rapid, 5-minute methods using our IS columns. Please look at the Waters webside for information!
How can I get a good peak shape for ketoconazole???... I've tested many different buffers, ph's and no success...
my mobile phase is ammonium acetate 10 mm + 1 mL of diethilamine / methanol
22:78
Column: C 18 250x4,6 X Terra
Is there any other better method you could suggest??
thanks
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Monday, May 3, 2004 - 03:29 pm:
We have run this compound without difficulty at alkaline pH on XTerra. What pH are you using? Alternatively, you can look at this application on Symmetry: http://www.waters.com/pdfs/Symmetry94.pdf
-------------------------------------------------------------------------------------------------------
By Carlos Teixeira on Tuesday, May 4, 2004 - 10:05 am:
Hello Anon-10:38;
X-Terra is a good (and roboust hplc-column), but, maybe you change Methanol by Acetonitrile? Wich Acn your chromatographic run will decrese (minor RTs). I beleave than with Acn your Ketaconazol peak will be better.
Good elutions!!
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 4, 2004 - 01:22 pm:
The problem is that i analyse creams and shampoo with no extra preparation, i just dilute and that's all..... but after a day of work i wash the column with water/meoh/acn/water and acn 30:70 for 2 hours.....
My x terra is new.... so that's why i'm surprising with the shape of the peak... I forgot to mention that when i chose this methodology, the peak was ok.....
the ph is 10
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Tuesday, May 4, 2004 - 04:34 pm:
I would think then that the problem is not that the column does not perform, but that something is accumulating on the column that your wash does not remove. Your wash is fundamentally OK, but maybe some assistance with the addition of acetic acid to your washes might help. I have to admit though, that this is a rather wild guess at what could be the problem.
-------------------------------------------------------------------------------------------------------
By Anonymous on Wednesday, May 5, 2004 - 07:28 am:
i'll try this one (acetic acid)
thanks!
-------------------------------------------------------------------------------------------------------
By syx_gf on Thursday, May 6, 2004 - 06:13 pm:
These days I work with ketoconazole shampoo. I use diisopropylamine (2 ml/1 L MeOH) - ammonium acetate (1 g/200 ml water)(70 : 30). By using ZE (Agilent) column I have a good peak shape. The problem is there are three peaks from our formula that elute too close to the ketoconazole's.
I think acid is not a good choice, it will be tailing. Ketoconazole shows good shape in basic mobile phase (pKa: 6.51 and 2.94).
-------------------------------------------------------------------------------------------------------
By Anonymous on Friday, May 7, 2004 - 08:19 am:
hi syx... I'm going to use acetic acid just to wash the column because of the tailing that my tests are presenting.....
but thanks a lot for your help, especially for an option for another column...
And by the way... what's the pH of your mobile using diisopropylamine? mine is about 10.
-------------------------------------------------------------------------------------------------------
By syx_gf on Sunday, May 9, 2004 - 06:12 pm:
pH of this mobile phase is higher than 9. The best pH for ketoconazole based on its pKa is above 8.51 (pKa+2). In this pH, we can not use "general" column, but a basic resistance column. I think X-Terra is OK for this condition.
-------------------------------------------------------------------------------------------------------
By lime on Monday, May 10, 2004 - 06:54 am:
Column : C18, 125 x 4.0 mm, 5 µm
Wavelength : 240 nm
Flow : 1.5
Inj. Vol. : 10 µl
Column Oven : 45 ºC
Mobile phase : 6 g ammonium acetate in 380 ml water.Add 320 ml methanol and 300 ml acetonitrile
try it. Its been validated for ketoconazole supp.
good luck.
-------------------------------------------------------------------------------------------------------
By Anonymous on Tuesday, May 11, 2004 - 07:44 am:
Hi lime...
i'll try this one...
Thanks a lot!!!
-------------------------------------------------------------------------------------------------------
By syx_gf on Tuesday, May 11, 2004 - 05:37 pm:
Finally, I decided to use diisopropylamine (2 ml/1 L MeOH) - ammonium acetate (1 g/200 ml water)(65 : 35)with gradient to higher organic conc after ketoconazole is eluted.
Column ZE (Agilent) C18, 5 um, 150 x 4.0 m
Flow 1.0
Temp 40
Work conc 40 ppm
Inj vol 20 uL
Rt Ketoconazole: 16.96', with Tf 1.18 and good resolution with our formula.
Lime, do you never have problem with your high conc buffer?
-------------------------------------------------------------------------------------------------------
By Lime on Thursday, May 13, 2004 - 07:13 am:
Hi !
Frankly, i have little problems with the column and i couldn't find out if it's about the mobile phase conc. or because we are working with supp. drug form.So, you might be right.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Sunday, May 16, 2004 - 08:39 am:
Lime: what is the pH of your ammonium acetate solution?
-------------------------------------------------------------------------------------------------------
By syx_gf on Tuesday, May 25, 2004 - 05:29 pm:
I think I should use SPE to eliminate other ingredients than ketoconazole. 45' total of my method is too long...
-------------------------------------------------------------------------------------------------------
By A.Mouse on Tuesday, May 25, 2004 - 06:13 pm:
What is your column length? Particle size? Why does it take so long?
-------------------------------------------------------------------------------------------------------
By syx_gf on Wednesday, May 26, 2004 - 12:40 am:
As I wrote on May 11. Sorry, Rt ketoconazole is 12.96, not 16.96. After 15' I use gradient to elute excipients behind the ketoconazole. (without gradient, they will elute until 90'!)
So, 30' behind is gradient section and re-equilibration.
-------------------------------------------------------------------------------------------------------
By A.Mouse on Wednesday, May 26, 2004 - 02:52 pm:
Then why don't you do a step gradient to the final conditions just after ketoconazole elutes? If you do not care about information in the remainder of the chromatogram, no reason to try for high resolution in a 30 minute gradient.
-------------------------------------------------------------------------------------------------------
By syx_gf on Wednesday, May 26, 2004 - 05:26 pm:
I stop the chromatogram after ketoconazole is
eluted. Then I do the gradient for 15 minutes, and reequilibration for 15 minutes as the formula: ((10 x Vm)+Vd)/F.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Saturday, May 29, 2004 - 10:43 am:
There are several things that you can do to speed up the method. After your last peak elutes, you can use a step gradient to elute the junk. The step gradient can be done at high flow. Since you are using a 15 cm 5 micron column, this should not be a problem. Also the reequilibration can be done at high flow. Also, 5 column volumes may be sufficient for reequilibration.
Also, you may be able to run your method at higher flow rate. Of course, you need to check, if the resolution of all peaks is still fine.
If there is not a problem at all, other things can be done as well. We have converted many lengthy methods to rapid, 5-minute methods using our IS columns. Please look at the Waters webside for information!