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- Posts: 4
- Joined: Sat Feb 11, 2017 3:04 pm
Thats my first post here. I have a few questions about LCMSMS analysis at basic conditions. I'm developing method for novel compounds new compounds that are synthesized in our company. At the "0" point of method development I check AUC in following conditions:
25 mM HCOOH in water (phase A) + ACN (phase B)
25 mM HCOOH in water (phase A) + MeOH (phase B)
10 mM NH4HCO3 pH 8.5 in water (phase A) + ACN (phase B)
10 mM NH4HCO3 pH 8.5 8.5 in water (phase A) + MeOH(phase B)
I'm using full gradient (from 5% to 95% B 2min)
Often I have to choose basic conditions with ACN, so I have few questions about these.
1) Now, I'm using Acquity BEH C18 column and still fighting with peak taling. I think about Eclipse Plus C18 column. According to the data sheet that column can work at pH range 2 to 9.
Is anyone working in these conditions with these column (or any analogue column)? I'm interested in column stability, reprodubility and lifetime in these conditions.
Generally, I wonder about the stability of the C18 column at a pH >7.
2) My compound degrades in basic conditions (0.1M NaOH pH 12.8 ) Can i observe these process during my analysis with NH4HCO3. I have not any data about compound in pH 8.5.
3) I want to use these conditions for plasma analysis.
Are there any contraindications or specific points on which I have to pay attention to?
4) At BEH AcQuity C18 data sheet I have read that Ammonium BiCarbonate is better (for column and MS detector), than Ammonium Carbonate, but why?
5) That question is not about basic conditions. I prepare calibration curve samples in plasma by spiking. What is the maximum amount of organic solvent (for ACN, DMSO, MeOH, EtOH and IPA) at plasma at which proteins will not be precipitated? I ask this because I'm still working with hard soluble (at water) compounds.
6) What is time for column equilibration? I have read that is 20 column volume. Now I'm using much less (about 8-10 column volume). I did not see any retention time drift. Is that correct column equilibation?
Sorry for terrible English