"Dilute and Shoot"

[kerri]

I _was_ trying to develop a quantitative method for a small drug using the 'dilute and shoot' concept. That is, perform a protein precipitation (from the biological matrix) using ACN , centrifuge, then take the supernatant and directly inject that on to the column. The problem: the two analytes and internal standard are piperazine derivatives and are polar (logPs: 1.35, 2.43, 2.07). I thought I might be able to overcome this problem by using a normal phase-inspired separation, e.g. Hypercarb or HILIC column and a high ACN or MeOH percentage. Well, boo on trying to be smart and develop the method this way. I wasn't able to achieve full resolution on the Hypercarb, though the peak shapes were ok.. On the HILIC, two compounds would co-elute (having symmetrical peaks) and the third is retained well with a very poor shape. Buffers don't help. They just impart bad peak shapes again.

I had worked on it for 6 days straight (with many different columns) and now I give up. I dont want to waste any more time and must move forward. Instead of being cute and getting my sample prep done in 20 minutes, I will just use the crash solvent, centrifuge, evaporate, and then reconstitute in the mobile phase (I know it's not bad, I just wanted to make my life even easier). I now have nice separation and peak shape on an Xterra using 20% ACN isocratically. I will continue my work this way.......

My question: has anyone successfully done this type of 'dilute and shoot' with a polar compound? At this point, I'm not going to change my method. .. but I can't get the question out of my head!

--edit-- Maybe 'dilute and shoot' isn't the best name for what I would like to do... possibly 'Crash and Shoot" is better hahahahhahahaaa

[Uwe Neue]

I do not see a problem with your method: evaporate, reconstitute...
Why are you concerned?

[kerri]

Right, the method is not a concern now.. This is just a time saving and interesting challenge and was looking to see if anyone had any more insight.

[HW Mueller]

If you check through this forum a little you will see that some people have been getting around mobile phase incompatibilities by injecting very small amounts of sample.

[Bryan Evans]

Some columns can handle this better than others - it's all about surface chemistry.
http://www.imtaktusa.com/site_media/fil ... TI450E.pdf

[pelle-plutt]

Instead of using ACN to precipitate the protein matrix, try using TCA (trichloroacetic acid). Add TCA to your solution to a final concentration of 6–10%. Most protein precititate at this concentration if the protein concentration is high enough (>0.2 mg/ml).
Of course, I don't know if TCA will affect your drug in any way...

[Baxthorpe]

I've done a few "dilute and shoot" methods for drugs, and one thing I can say is that you will clog the top frit/mesh or injector filter very quickly if you do not sample using an inline filter.
You can get a luer fit stainless steel filter holder and GF/F filters from Whatman, suck the sample through it into a syringe, and you won't need to watch the back pressure, or get the spanners out every 50 injections...

You might have better luck on the hypercarb if you try to run the samples as ion-pairs - add, say 0.005% formic acid to the eluent, and the interesting substances will form ion-pairs with the acid. Then they will probably stick there on the column "forever" until you add a tiny amount of an amine (eg. diethanolamine) into the eluent to compete with them for the retention sites. Methinks this may be why pelle-plutt is suggesting TCA.

There is also the consideration that a small sample, as suggested above, can improve the apparent resolution of a poor separation. Of course, then you have to go more sensitive at the detector, and the noise goes up ...

[HW Mueller]

Because of Baxthorpe´s contribution I noticed that my statement above was not clear, it was supposed to refer to small volume injections, not small analyte amount. Small volumes often do not produce effects due to solvent-mobile phase incompatibilities.
Also, for pelle-putt: If you use TCA for the precipitation you have a very good chance to coprecipitate hydrophobic analytes. That´s why methods with precipitation by organic solvents were deviced, they keep such analytes in solution. Example: Cortisol.

[kerri]

All of your input is greatly appreciated! Thank you!!


My failed results came from injecting 5uL on a 2*50mm column. I might just try lowering that amount today to see if that helps.

Unfortunately, TCA and TFA both cause considerable ion suppression (at 0.05%) so TCA precipitation is out (using LC-ESI-MS/MS). I even tried ion pairing (being sure to bypass the degasser) with a perfluoro acid and that just caused major tailing. Tried ammonium formate buffer pH 4 and also got tailing.

Baxthorpe- I tried DEA with the hypercarb (according to the hypercarb method development guide) and it caused two of my three compounds to be retained forever. I had to use 0.1% formic acid to free them.

I do use a precolumn filter/frit to protect the column from particulates.

HW Mueller- Thank you for the specific drug examples. Trying to find the right terms to search in SciFinder is challenging!!

I am still in the beginning stages of this method development..simply because I am waiting for a replacement internal standard. So I can try to optimize this time saving method.

[dr_Pyrex]

I think this concept can be useful for you in the "fight" for better S/N:
http://www.sigmaaldrich.com/analytical- ... e-ppt.html
it is very easy and can be easy add to "dilute and shoot" idea.

[Camisotro]

I don't suppose it's possible to 1) reduce the volume of ACN used for the crash, and then 2) dilute the ACN phase down to less than 20% with water? That way you will still be injecting your sample in a more polar solvent than the mobile phase.

[Baxthorpe]

It is sometimes hard to take me seriously, even for me!

Let me be clear, I have not been a practising chromatographer for about 10 years, I make my living elsewhere these days, and it is only by serendipity that I was drawn to this forum and made a few posts.

OTOH I have knocked up a few methods that were included in pharmacopoeias, and successfully challenged pharmacopoeal methods in the past.

I don't like to boast, but I think you need to know that this old fart is not just some machtmensch trolling. A few posts is not much to work on, and
sometimes I say the entire opposite of what is needed - but keep digging, that's just me misunderstanding things. And not being sufficiently clear.

HW Mueller - no need for clarification - I understood that you were suggesting that a small volume injection would reduce the solvent "difference" interactions. You merely reminded me of a separation I did about 25 years ago, when 4 sharp peaks on a 0.5 microlitre sample would transform into a drawing of a stegosaurus using a 20 microlitre loop ... I only wanted to point out that a small sample can give a nice chromatogram in addition.
Not so useful for drugs work, in any case, because that sort of thing can allow decomposition products to limbo-dance under the detection limits. Not that it matters here ... if the dilute and shoot is to succeed, there is only one permissible sample concentration.

Kerri - your Hypercarb results offered promise, you didn't realise how good they were. One component of three eluting is GOOD, if they are similar compounds - it says you are close, you just need to get closer.

You have also determined two additives that can control your separation.

But you did not understand my musings. Ok I got something backwards again - I get my mords wuddled regularly. So I'm just going to say what I would do, then let you dig the bits out you need.

If I were looking at this separation, I would use an A/B gradient pump with A running eluent + 0.05% formic acid, and B running the same eluent + 0.07% diethanolamine.
Running at 100% A would have everything whizzing through, so go for 90% A / 10% B for a start, then adjust depending on what happens.

Poor separation, low retention, increase the proportion of B. Vice versa, decrease B. Get the needed separation, but retention time is unsuitable, adjust the major organic component content of the eluent. Muck about with it until you have a suitable system.

When you have found an appropriate A/B ratio, you should tune the method by running that mix as "A" against a B eluent without the formic acid and diethanolamine, to determine a low concentration which will give consistent results repetitively. Go low, and the retention times will "walk about" with each consecutive injection. Go below that, and some very strange things will happen.
To be honest, you don't really need the last bit on this separation if you have it working. But if you are using straight chain sulphonic acids, the tuning step can save a lot of money.

There is also the consideration that the eluent cannot be stable (it is good enough for a day's work, but after a few days it is jungle juice). From experience, you can work these sort of solutions from yesterday afternoon into this morning if you chill them overnight, but definitive work should always be performed on freshly prepared eluent.

LOL who said it was easy ...

[kerri]

Diethylamine? or Diethanolamine?

I had tried Diethylamine previously... I might have to ask our neighbors if they have any Diethanolamine.

All of your knowledge is helpful!!! I now have enough retention on the Hypercarb.. just tailing peaks (up to 0.25% formic acid). So I will get right on your superb suggestion Baxthorpe (can you believe I am the first one in my lab to even care to try such a 'crash and shoot' procedure!)..

I have a renewed spirit thanks to all of support in this forum.

When I return to the lab in the morning I will post the latest chromatogram...

Cheers!

[kerri]

Here is the best assofar:

-Piperazine derivatives (all similar)
-Hypercarb 2.1x50mm 3um
-5ul of 500 ng/mL (each compound) in 100%ACN + 0.1% FA loaded
-200uL/min 50% ACN + 0.2% FA isocratic

SO if I can get these peak shapes all sheared up, this 'crash and shoot' method will work well! Otherwise, I will stick to drying, the reconstitution and loading on either a T3 or Xterra.

[url=http:/imageshack.us/photo/my-images/246/hypercarbsep.jpg/][/url]

[Stryder08]

Here is the best assofar:

-Piperazine derivatives (all similar)
-Hypercarb 2.1x50mm 3um
-5ul of 500 ng/mL (each compound) in 100%ACN + 0.1% FA loaded
-200uL/min 50% ACN + 0.2% FA isocratic

SO if I can get these peak shapes all sheared up, this 'crash and shoot' method will work well! Otherwise, I will stick to drying, the reconstitution and loading on either a T3 or Xterra.

BZP, mCPP, and TfMPP?