Chromatography Forum

Anna: The idea is to bind more of the phospholipid by interaction with the polar function of the packing while still eluting the analyte successfully. A 50/50 mix of acetonitrile and methylenechloride is a reasonable suggestion, not knowing anything about the functional group and the possible retention behaviour of your analyte. If you go too hydrophobic with the mobile phase (for example pure methylene chloride), it is possible that your analyte will not elute either.
Hans: this is a two-dimensional method, maybe even three-dimensional... Off-line, of course...

I get that, Uwe. We are working on uElution plate with 25uL eluate. Could we inject 2-5uL ACN:methyleneclorid 50:50 directly in the LC without evaporation/reconstitution in mobile phase? I've heard methylene clorid can attack PEEK tubings. It's not not miscible in water, but may work with low injection volume and 50:50 MeOH:H2O?
(Obviously, there might be a serious mismatch with the mobile phases and bad peak shapes).

Low injection volumes might be OK, but I still think it is better to evaporate MeCl2. It has a very low boiling point (40 degrees Celsius), so it will disappear faster than you can look.

Ok, but it sounds like a cartridge type of device is involved. I was thinking of something more sophisticated, maybe a good precipitation/extraction or ultrafiltration, with the first chromatographic step being done with a full sized internal RP size exclusion column (seems I got the description wrong?).

Solid-phase extraction – properly carried out – is actually a fairly sophisticated technique. After we introduced Oasis, my team created immediately detailed procedures that relied on the simultaneous use of 2 dimensions – reversed-phase and pH – to optimize extractions. Then later on with the introduction of mixed-mode packings, 2-dimensional procedures based on reversed-phase and ion-exchange became the standard. The latest elution schemes are still more advanced uses of the same principle, with emphasis on practical simplicity.

Properly carried out SPE is not voodoo, but clever use of the chemistry tools at hand.

But it usually exploits stark differences, it´s sort of sophisticated rough stuff, for fine tuning one uses a longer column. If I had trouble with a SPE I would have, in the time of this discussion, long since tried to add another "normal" chromatography and/or start to revamped the cleanup. Actually, a look at lipid methods before MS was discovered by chromatographers might provide a super solution. Such an "old" method, if it exists, + MS should be neat.
(This high throughput, etc., etc., has been a high money transfer wherever I have looked).

I have tested ACN:methylenclorid (1:1) as elution solvent, evaporated and reconstituted. Most importantly the matrix suppression was reduced to about 10% (n=4). The amount of phospholipid is less, but have not fallen dramatically. Recovery of the analytes is about the same.

Perhaps I should investigate even higher concentrations of metylenclorid? 75%?

As I mentioned, could it be an idea to to NH4OH washing of the sorbent? Perhaps something like this?

Load sample
Wash with neutral H2O
Wash with 5% NH4 in X% MeOH
Wash with H2O with 2% formic acid
Elute

Could this help to remove phospholipids during the wash step? Remember, I am not interested in any basic substances, i actually want to remove them. By washing with H2O 2% foac before elution, I ensure that they are locked to the sorbent by ion exchange.

We apparently have moved into the right direction by using a more hydrophobic solvent to leave more of the phospholipid with its very polar head group behind. This is promising. However, a further increase in methylenechloride may create a bi-phasic system, which would make a mess out of the entire idea. It is worth exploring, but don't be surprised if you get junk.

I am not sure that polar washes will help a lot, since phospholipids are rather hydrophobic, and the fact that you got coelution on your reversed-phase chromatography indicates that an increase in the organic content in your polar wash will not be beneficial.

Phospholipids are supposed to have a reasonable solubility in chloroform. Maybe a substitution of the methylenechloride with a lower fraction, maybe 25% of hexane in 75% acetonitrile will do a better job. Again, hexane evaporates readily, same as methylenechloride.

Is hexane miscible in acetonitrile? I'll find out tomorrow...

I tried with MeOH:Hexane 75:25, but that didn't improve anything. Matrix effects increased to about 20-30%. (Substance added to reconstitution solvent-method). This is comparable to what I've seen with 100% MeOH elution.

In parallel I ran the same samples eluted with ACN, and they showed a 10% suppression of their signals.

The chromatograms from MeOH:Hexane elutions indicated higher levels of phospholipids than when eluted with ACN alone.

MeOH and Hexane have mixing problems, which is why I had suggested to use MeCN and Hexane. Plus I want to move away from a hydrogen-bond donor.

Acetonitrile and hexane are not miscible. I looked it up and even tried it.

You are right. I just checked: acetonitrile and hexane are not miscible. I am surprised since I got this (theory) publication that states the opposite. Oh well...

But methanol and hexane are not miscible either, plus it did not work for you. Where does this leave us? Maybe your original suggestion of a higher methylenechloride concentration in acetonitrile is the way to go. The attempt is still to play on solubility of your steroid while leaving the phospholipid behind.

Supelco recently supplies a special SPE cartridge called HybridSPE which selectively use for removal of phospholipid from plasma sample. I am trying this at the moment and would let you know the results later.

Acetonitrile and hexane do not mix in all proportions but you can get a fair bit of hexane to dissolve in acetonitrile. Remembering back this was the basis of a pesticides in fats clean up method, but the details escape me now.

Peter