Chromatography Forum

Hello everybody

I am working with 4 polar compounds that does not have retention in chromatographic column (C18 and C8 from Jones, Agilent, Luna, Gemini).
As I am extracting them from plasma samples, even if I use a mobile phase 50-50 Acetonitrile-ammonium carbonate or acetate (1mM or 1.5mM) there are matrix effect at the same retention time of my compounds (at least one of them).

I am using liquid-liquid extraction (ethylacetate-hexane-diethyl ether or dichloromethane-methyl terc butyl ether), but non of them decreased the suppression.
Does anybody have a suggestion on a different mobile phase or modifiers ???????
Best regards.

Maristela,

What is the nature of your polar compounds: ionic or non-ionic?
What is pH of you mobile phase? If your compounds are hydrophilic you will have hard time retaining them on the reverse phase column. You can try either HILIC separation or mix-mode separation. I am not an expert in HILIC, but we have few people on this board.

Mixed mode will work if you have ionizable hydrophilic, hydrophobic ionizable compounds (amines, amino acids, organic acids). They will be retained by ion-exchange mechanism.

Check the following links may be they will help you:

http://www.sielc.com/pdf/SIELC_June_2005.pdf comparative examples for polar compounds)

If you have acidic compounds you can use "Direct Plasma Approach":

http://www.sielc.com/Technology_DirectP ... lysis.html

regards,

Vlad

Maristela,

What I do not understand is that you talk about polar compounds that do not have retention in reversed phase material and then you say that you are using 50:50 ACN:H2O and that you observe matrix effect at the same retention time of your compounds (at least one of them) which means that your compounds are separated one from another?

If these compounds did not have any retention in C8, C18 they would have been eluted all together in the void volume at 50:50 ACN:H2O. Have you tried gradient elution from 0% ACN to 50% ACN?

What is the retention of your compounds in high aqueous conditions?

see http://users.chartertn.net/slittle/ , first topic on page which talks about some of my experiences with matrix effects.

Acetonitrile doesn't do a good job of getting lipids off columns. Could cause you some problems.

I like mixtures of MeOH and acetonitrile, again see

http://users.chartertn.net/slittle/ first topic on page..

you didn't say which anticoagulant you were using in the blood collection.

Some of the heparins might cause problems since polymeric in nature. Probably better using Na EDTA or K EDTA..

Did you inject the plasma while injecting the analytes post column to see if can find a definitive component or components causing matrix effects.

good book with matrix effects

http://www.amazon.com/gp/reader/0849319 ... eader-link

Using Mass Spectrometry for Drug Metabolism
Studies, CRC Press, Boca Raton, FL

Good luck, matrix effects can be very bothersome..

Dear Vlad
Thank you for replay.
I am analyzing 4 ionic compounds (losartan, valsartan, hidrochlortiazide and clortalidone), The pH of the mobile phase is around 6-7. I tested pH 3, but there was a decreased od the response.
As they do not have retention in the column, they are in the void as all the matrix compounds. I have a separation between the losartan/valsartan and hct/clortalidone, but the supression is in the beginning.
I was thiking in a mobile phase modifier that could push my compounds a little bit further, in other words, increses the retention time and doing so, the supression stayed before them.
Maristela

Maristela,

What is the nature of your polar compounds: ionic or non-ionic?
What is pH of you mobile phase? If your compounds are hydrophilic you will have hard time retaining them on the reverse phase column. You can try either HILIC separation or mix-mode separation. I am not an expert in HILIC, but we have few people on this board.

Mixed mode will work if you have ionizable hydrophilic, hydrophobic ionizable compounds (amines, amino acids, organic acids). They will be retained by ion-exchange mechanism.

Check the following links may be they will help you:

http://www.sielc.com/pdf/SIELC_June_2005.pdf comparative examples for polar compounds)

If you have acidic compounds you can use "Direct Plasma Approach":

http://www.sielc.com/Technology_DirectP ... lysis.html

regards,

Vlad

Dear Kostas
Thank you for the replay.
My compounds have a little separation, that does not have an importance for me. What I want is to separate them from matrix compouns, that most of the time are in the beggining of the run (in the void of the column)
I was thinking in one mobile phase modifier that could increase the retention os the compouns in the column, separating them from the supression.
I tested gradient, but I should increase the run time. I would like to find another alternative.

Maristela

Maristela,

What I do not understand is that you talk about polar compounds that do not have retention in reversed phase material and then you say that you are using 50:50 ACN:H2O and that you observe matrix effect at the same retention time of your compounds (at least one of them) which means that your compounds are separated one from another?

If these compounds did not have any retention in C8, C18 they would have been eluted all together in the void volume at 50:50 ACN:H2O. Have you tried gradient elution from 0% ACN to 50% ACN?

What is the retention of your compounds in high aqueous conditions?

Maristela,

Based on the structures of your compounds you should not have any problems retaining losartan and valsratan. I expect the retention to be at least 10 min on 150 mm column at 50/50 ACN-water due to good hydrophobicity (couple benzene rings, alkyl chain, etc.).
Chlortalidone and hydrochlorthiazide a less hydrophobic or rather hydrophilic and you need a gradient to have reasonable retention for all four compounds. Going from 10% ACN to 70% might help you.

Hydrochlorothiazide has aniline fragment in and you can use weak basic properties of it to retein it by combination of ion-exchange and RP properties. For this purpose you can use Primesep 100 column.

here is application for some chlorsulphonamide structures similar to chlortalidone and hydrochlorthiazide

http://www.sielc.com/compound_084.html

I would advice to use a guard column to prolong the life of your column.

Contact us if you have questions,

regards,

Vlad

Vlad

I tested a NH2 column (Alltima NH2 - 3um 150 mmx 4.6mm), but the MS response decreased a lot in spite of the retention.
This is a mixed mode column and I tested acetonitrile:water, acetonitrile: (NH4)2CO3 1mM, acetonitrile:ammonium acetate 1mM, but there is no peak to losartan .

What kind of mobile phase should \I use with this kind of column ???
Maristela

Maristela,

Based on the structures of your compounds you should not have any problems retaining losartan and valsratan. I expect the retention to be at least 10 min on 150 mm column at 50/50 ACN-water due to good hydrophobicity (couple benzene rings, alkyl chain, etc.).
Chlortalidone and hydrochlorthiazide a less hydrophobic or rather hydrophilic and you need a gradient to have reasonable retention for all four compounds. Going from 10% ACN to 70% might help you.

Hydrochlorothiazide has aniline fragment in and you can use weak basic properties of it to retein it by combination of ion-exchange and RP properties. For this purpose you can use Primesep 100 column.

here is application for some chlorsulphonamide structures similar to chlortalidone and hydrochlorthiazide

http://www.sielc.com/compound_084.html

I would advice to use a guard column to prolong the life of your column.

Contact us if you have questions,

regards,

Vlad

James

I am more interested in separate the lipids from my compounds than avoid them. I tested several solvents for the plasma extraction and I have two of them that give me a good recovery.
The anticoagulant is heparin.
I would like to separate my compounds from supression region. I tested C18, C8 (Jones, Gemini) and Alltima NH2. These last one has a good retention for one of my compounds but the other " desapeared", in othewr words it does not elute neather in 70% water nor 70% of acetonitrile.
I tested some mobile phase modifiers as TEA, (NH4)2CO3 and ammonium acetate 1mM, but with no improving in the results.
Maristela

you didn't say which anticoagulant you were using in the blood collection.

Some of the heparins might cause problems since polymeric in nature. Probably better using Na EDTA or K EDTA..

Did you inject the plasma while injecting the analytes post column to see if can find a definitive component or components causing matrix effects.

good book with matrix effects

http://www.amazon.com/gp/reader/0849319 ... eader-link

Using Mass Spectrometry for Drug Metabolism
Studies, CRC Press, Boca Raton, FL

Good luck, matrix effects can be very bothersome..

I agree that your problem can be solved with a gradient, and that you do not need to wait forever.

I suggest to use a 2 cm 2 mm column, 3 or 5 micron is fine. Use a C18. Run a gradient at 1 mL/min from 0 to 80% acetonitrile over maybe 2 minutes. Let your system run a bit longer, since you may a substantial gradient delay in your system.

The sample should be injected in water. You can evaporate your organi sample to dryness and redissolve in water.

A lot of the ion suppression will be gone, especially if you stick with your liquid-liquid extraction.

For this trick to work best, you will need to use the LC conditions pretty much as suggested.

If you want to know if you elute the lipids from the column and separate them from the drugs of interest, try my method at

http://users.chartertn.net/slittle/ under the matrix effects section.

easy to do and you will know for sure where they elute.

Acetonitrile by itself does not elute the phospholipids very well at all (very hihly retained in this solvent, and broadly eluted).

Either pure methanol or up to 75% acetonitrile in methanol works well. I normally use 50/50 for most things, but other compositions offer some advantages depending on the analyte.

I found the Monochrom C18 (details of some separations on my website, above) column very highly recommended in the literature by someone who did a comparison of a variety of columns and have used it for all my drugs in plasma work.

Should definitely use a gradient. Again, one example on my website.

Never tried heparin as anticoagulant, but heard since it is polymeric in nature, can elute in many places in the chromatogram and cause problems. I have used EDTA (Na or K).

I have a paper in press on more aspects of this work. I found if you don't elute the phospholipids they begin to breakthrough. When they breakthrough, if your one of your analytes elutes at that point, its response in positive ion will change. After 5-6 analyses yield a steady-state response and the analyte response is again constant.

However, works best with unlabeled materials when use gradient. Isocratic not so good.

The lipids will probably be in your sample in differing amounts depending on the solid phase extraction or liquid/liquid extraction method used. I usually just precipitate the plasma with acetonitrile using a method I found in the literature. I think the reference is also on my website.

Good luck. Aggravating work..