how to develop a lc-ms/ms method for acidic compounds plasma

[GURU]

please explain follwoing what are the thumb rules (basics) for developing method on LC-MS/MS for bioanalytical purpouses?????

1. how to develop a lc-ms/ms method for acidic compounds in human plasma?

A. how to select a buffer for acidc compounds
B. how to select a mobile phase for acidc compounds
C. how to select a Extraction procedure for acidic compounds
D. how to select a column for acidc compounds
2. how to develop a lc-ms/ms method for basic compounds in human plasma?

A. how to select a buffer for basic compounds
B. how to select a mobile phase for basic compounds
C. how to select a Extraction procedure for basic compounds
D. how to select a column for basic compounds
3. how to develop a lc-ms/ms method for neutral compounds in human plasma?

A. how to select a buffer for neutral compounds (zwitter compounds)
B. how to select a mobile phase for neutralcompounds
C. how to select a Extraction procedure for neutral compounds
D. how to select a column for neutral compounds

[Uwe Neue]

For compounds with a basic functional group (bases or zwitterions), you get typically the best sensitivity at acidic pH, using formic acid as the mobile phase additive and positive ionization mode. If your chromatography is not what you want (overlap with matrix interferences), you can still use an ammoniumbicarbonate buffer at pH 10. You may loose a factor of 3 or so in sensitivity, but if this removes matrix interferences, then your assay sensitivity may actually improve.

For acidic compounds, you can get the best results in negative ionization mode with ammoniumbicarbonate at pH 10 or ammonia. For zwitterions where you have the option of positive or negative ionization, the positive ionization mode is typically preferred (higher sensitivity).

You need a suitable column such as XTerra or XBridge to work at alkaline pH in the mobile phase. These packings can of course also be used under acidic conditions, but most modern columns based on a high-purity silica or a hybrid packing will do fine at acidic pH.

I am a very strong advocate for solid-phase extraction prior to LC/MS. You will get much cleaner backgrounds (orders of magnitude!) compared to protein precipitation. It is a bit more work, but there are well established generic methods that have been developed (look for the Oasis applications booklet on the Waters website). The overall best results are obtained with mixed-mode ion-exchangers. A generic method for an amine works as follows:

1. Acidify your plasma sample and add internal standard.
2. Load this sample onto the Oasis MCX (mixed mode cation exchanger) cartridge
3. Wash with aqueous acidic solution
4. Wash with methanol
5. Elute the basic or zwitterionic analyte in methanol with ammonia


For acidic analytes, change the pH scheme and use the mixed-mode anion exchanger. Additional finetuning can be done, if the results are not yet satisfactory, which is rare.

When you develop the method, collect all fractions and look for you analyte and internal standard in all fractions, so you understand what has happened to it. Once you have a functional method, you only need to analyze the fraction with the analyte.

If you contact me, I can give you several publications that describe the backgound for these methods.

[alpesh]

i would like to suggest you to go through all waters catlogue. it has solid phase methods for all kind of compounds, either acidic, basic or amphoteric. column and mobile phase selection is very practical. one can hardly predict the mobile phase and column by simply looking at structure or other like properties of the molecule. you have to practically work on particular molecule.

[promochrom]

Hi Guru,

You may have a look at Primesep D column from SIELC for direct analysis of acidic compounds in Plasma. It is a mixed mode column with both reverse phase and anion exchange function. When the pH of mobile phase is < 3, all the proteins are positively charges and are excluded from the column withour retention. The neutral and acidic compounds are separated by the reverse phase mechanism.

You may find more information from SIELC or from the catalog (page 24) from following website :

http://www.gc-lc.com/Catalog_0809US.pdf

PromoChrom

[GURU]

1. what i mentioned below for bioanalytical method development predictions were correct or is there any changes. please explain if any tips were there for better clarification????

2. On what basis buffer pH has to adjust (based pka of drug or based on pka of buffer or both things has to consider) please explain me with better examples?????

1.Genaral extraction procedure:-

If a compound is polar solid phase extraction was prefferable
If a compound is non-polar or moderate polar liquid-liquid extraction was prefferable
If a compound is highly protein binding protein ppt was pfrefferable

2.Buffer selection:-

for acidc drugs:- adjust the ph of buffer +/- 1.5 ph units pka of an compound i,e basic ph
for basic drugs:- adjust the ph of buffer +/- 1.5 ph units pka of an compound i,e acidc ph
for acidc drugs:- adjust the ph of buffer +/- 1.5 ph units pka of an compound i,e basic or acidc ph

Column selection:-

for polar drugs :- for acidc drugs C18,C8 and for basic drugs cyanide(CN) columns were prefferable.

mass spectrometric mode selection:-

for acidc drugs -ve mode was prefferable
for basic drugs +ve mode was prefferable

[Uwe Neue]

2. The ionization of analytes for MS detection is solely a question of the detection. There are often contraditions between the requirements of chromatography and MS, but for most cases you can get decent results by following the rules that I laid out in my last post.
In addition, you do not have a lot of buffer options in MS. TFA is a general good additive, but it creates ion suppression on its own. Formic acid is better. Acetic acid would work too, but you are better off with formic acid or formic acid buffers. In the alkaline pH range, you want to use ammonia or (better) ammonium bicarbonate. There is not a functioning MS compatibe buffer around neutral pH.
You need to be more concerned whether your analytes will form easily positively charged or negatively charged species for MS detection. This is the primary factor for your buffer selection. pKa is insufficient information.

1 Extraction procedure:
solid phase extraction will work for any analyte, from polar to non-polar. Liquid-liquid extraction works best, if your analytes can be put into a non-ionic form. You use strong acid or base for breaking the interaction of the analyte with protein before you do SPE or LLE. Protein precipitation has always in every one of our comparisons given inferior results to SPE or LLE. It is by far not as good as the alternative methods.

2, buffer selection: I believe that I have clarified above what your buffer options are. The concern about the pKa of your drug is irrelevant.

3. Column selection: I would never use a CN column, especially not in front of an MS, unless I am interested in doing a lot of cleaning of the source. In addition, for polar drugs, CN gives less retention. For polar drugs I recommend a C18 that is compatible with 100% water (T3 from Waters), or HILIC. HILIC gives better MS response.
Otherwise, I recommended pH choices and column choices in my first e-mail.

[yangz00g]

Guru,

Your questions are too broad and the answers are hard to be summarized. I use acidic compound to show you my strategy for a method development

In order to develop a suitable LC-MS/MS for acidic compounds, you first have to know what kind of method you want. For MS/MS detection with isotope labeled internal standards, baseline separation is no longer necessary, the main function of a LC column is to retain the compounds for a certain period of time so that they can avoid ion suppression region. To select a suitable column , I will look at the Pka and logP vlaue of the compound. If the compound has a small log P (e,g. logp = -2) and Pka value (e.g. Pka=2), C18/C8 is definitely not a good choice, because the compound is too polar, and no MS compatible buffer can give you pH~1 and most silica based column doesn't work in that pH range. As Uwe mentioned, HILIC may be a better choice at this point. Once I pick the column, say the HILIC, next step to select the mobile phase, a quick literature search will give you a starting point to optimize the mobile phase/buffer components. For plasma samples, a protein precipitation followed by SPE will be my way to treat the sample in order to futher eliminate ion suppression,

If you do need a baseline separation of all you compound, play with mobile phase components to get the desired separation.

Comparing to LC, MS/MS optimization is relatively simple, a compound dependent parameter optimization will help get the sensitivity you desire.

Hope this help.

[GURU]

It's a gr8 pleasure and all heart fully thanks to Uwe Neue and yangz00g for answersing my quiries. I have lot of basics quires regarding bioanalytical method development. if u dont mind shall you (Uwe Neue and yangz00g) answer my questions clearly for better understanding.shall i post my quires to you daily?

[GURU]

1.when back extraction was prefferable for extraction of drugs from plasma, how back extraction will be done and which type of compounds will be go back extraction ?
2.Whats the difference if carbon load was less or more in column how it will effect on LC if carbon load is less or more?

please explain follwoing questions Elaborately with examples:-
1..What Functional goups will be present if compound is in acidic nature explain this elobaorately?
3.What Functional goups will be present if compound is in basic nature?
4.What Functional goups will be present if compound is in neutral?
5.mechanisum action involved in quadropoles (how ions will separate in quadropoles)

[Uwe Neue]

1. I do not know what "backextraction" is referring to.
2. Carbon load is the amount of carbon per (silica + ligand). It is not a useful measure for retention nor for other things. Unfortunately, it has become a tradition to report such values.

1. The common acidic functional group is -COOH, but it could also be -SO3H. Weakly acidic functions are phenol or sulfonamides.
3. Basic analytes commonly have a nitrogen attached purely to neighboring aliphatic carbon or hydrogen. Weakly basic functions are aromatic amines or nitrogen in a ring.
4. most other functional groups represent neutral molecules -OH, =CO, -NRCO.

These rules above are rough. If you have a conjugation through an aromatic system between different functional groups, you can be in for some surprises. For example, caffeine essentially is a neutral compound.

I leave the quadrupole question the an MS expert.