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We're doing CYP 2D6 inhibition assays inhouse using bufuralol as the
substrate. I am at my wit's end trying to develop a method for the
analysis of hydroxybufuralol by lc/ms/ms. All the C-18 columns I've
tried do not seem to retain the analyte and elutes in the void or very
close thereafter. I've tried running a ballistic (<1 min) gradient
from 5 - 95% organic (0.1% formic acid in acetonitrile), a shallow
gradient (ca. 5 min), and lengthening the initial conditions of 5%
organic to about 1.5 min to no avail. Interestingly, the last column
gave me a chromatogram in which significant amounts of
hydroxybufuralol eluted < 1 min but some then eluted around 3 min. I
am completely baffled by this. Anyone have any experience with this
compound from hell?
Thanks in advance!
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The following message was posted to: PharmPK
Try the following:
Varian Pursuit Diphenyl 3u 50x2mm
0.7 ml/min
A=water 0.04% FA; B=ACN 0.04% FA
Gradient:
Time Module Events Parameter
0.01 Pumps %B 17
0.30 Pumps %B 17
0.40 Pumps %B 35
1.50 Pumps %B 50
1.60 Pumps %B 99.0
1.61 Pumps %B 1.0
1.90 Pumps %B 1.0
2.00 System Controller Stop
We use 278/159 for the MRM. The retention time should be about 0.8
minutes.
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The following message was posted to: PharmPK
It sounds like you have too much solvent in your samples, resulting in
premature elution or split peaks. If you have just done a protein
precipitation, you might have 80% acetonitrile in the sample. First, try
adding an equal volume of water; inject the same volume, don't double
it.
Regards.
Ted
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The following message was posted to: PharmPK
Dear Tai Ly,
In CYP study you will get lots of unwanted matrix that can hamper your
molecule of interest. Hence you have to choose your gradient method
(chromatography) very accurately to avoid such unwanted junks. You
have to be very careful about your late eluting peaks also.
You can try the below mentioned condition. You will definitely get a
peak around 1.7 minutes.
Mobile Phase:
A: Acetonitrile
B: Ammonium Formate (5 mM with 0.125 % Formic Acid)
Gradient Method
Time A% B%
0.01 25.0 75.0
1.60 90.0 10.0
3.00 90.0 10.0
3.10 25.0 75.0
5.00 25.0 75.0
Mobile phase flow rate: 800uL/min.
Three way splitter: 80% out
Mass Fragment (m/z): 278.00/186.00
Column: Chromolith Speed ROD, RP-18e, 50-4.6mm
Regards,
Susipta Basu
Senior Research Scientist (DMPK-Bioanalytical)
Sai Advantium Pharma Ltd.,
Pune-57
E-Mail : sudipta.b.aaa.saiadvantium.com
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