Chromatography Forum

Hey all,
Good morning,
I am trying to quantitate few groups of lipid molecules. Before starting I want to get suggestions from experts in this forum.

1) Can I use precursor ion scan for quantitation (For example 184 precursor ion scan for phospholipids, sphingomyelins?)
2) What is the best way for quantitation (full scan, precursor ion scan, product ion scan, MRM)? Can these scans be equally used for quantitation?
3) As I see people spike internal standard and quantitate with reference to internal standard? However I have not see people reporting about matrix effect. My question is- Can I spike internal standard to my sample and use the spiked internal standard to quantitate without knowing the matrix effect? I think some amount is lost during extraction and processing?
Please suggest me the best method for quantitation (if there is stepwise protocol for any compound that will be great)?

4) What can be the placebo matrix to measure matrix effect for human plasma sample?
5) Is the below formula the only method for quantitation?

For example:

Analyte: 40000 (Peak intensity)
Internal standard: 60000 (Peak intensity)
Conc of internal standard: 1ng/ul

Response factor = Intensity of internal standard/Conc. of internal standard
= 60,000/1
= 60,000

Conc. of analyte = Peak intensity of analyte / Response factor
= 40,000/60,000
= 0.666 ng/ul

Is this the correct way of quantitating unknown (analyte) with reference to internal standard?

Please help me by answering my queries. I seriously need to get a very reliable and precise method of quantitating unknown using internal standard.

Thank you.

Sirus

The formula I have always used, and most MS data systems will use if you tell them you are using an internal standard is

RF=(At * Cis)/(Ais * Ct)

Where
At=Area of target
Ais=Area of internal standard
Ct=concentration of target
Cis=concentration of internal standard

You apply this to a calibration standard containing both the target analyte and internal standard to get a Response Factor

Then you solve for Ct and use the area of the target, area of internal standard and concentration of internal standard in the unknown sample to find the concentration of the target analyte in the unknown sample. The data system for the instrument should have this already built into the program.

Internal standards can be added before extraction to compensate for extraction efficiency, instrument drift and matrix effects or they can be added after extraction to the final sample prior to analysis to compensate only for matrix effects and instrument drift. I prefer to add just prior to analysis, and use a matrix spike sample to judge the extraction efficiency, where you spike a known amount into a duplicate sample and calculate the recovery after subtracting the amount of the unknown sample from the matrix spike final concentration.

The formula you posted only works if the instrument response for the internal standard is the exact same as for response for the unknown. You can get close with isotope labeled versions of the actual component, but even those have some variation in instrument response for a given amount of analyte present. It is useful for screening but not for finding exact concentrations.

James, do you have literature pointer for this equation?
Thanks
Jörg

James, do you have literature pointer for this equation?
Thanks
Jörg

Before James replies see on page 18:

https://www.epa.gov/sites/production/fi ... -524.2.pdf

I took my info from EPA SW846 Method 8260, but should be the same as the Method 524. Most all of the EPA chromatography methods reference this equation.

Quite unfortunately internal standards do NOT work in LC-MS the same way that they do in GC-MS. The ionization mechanism is completely different, and this greatly complicates the use of an internal standard. If you have a deuterated analog of the compound of interest you probably will have some success, but otherwise it is problematic.

You are much better off using the method of known addition. Spike at 2 or 3 concentration levels and you'll be able to determine the original concentration of the analyte in the sample. This will compensate for matrix effects in the sample.

We typically use 2 ions with single quad MS, so we look for the molecular ion (your "precursor" ion) and the most intense fragment. You can vary the degree of fragmentation on most single quad MS systems. I am not sure which system you are using, so you'll need to look at your documentation to see how to achieve this.

We quantify in selected ion mode (MRM). It seems to be more stable and gives fewer interferences than full scan mode. I am running an Agilent 1100 MSD (1946D) with ESI.

Make sure that your mobile phase is compatible with the MS (avoid TFA) and make sure you include enough available ions in your mobile phase (like 0.1% acetic acid, ammonium acetate, etc). The ionization is very soft, so optimum conditions have to be experimentally determined.

Thanks for helpful suggestion and information all.

In addition,
How is resolvation volume included during quantitation? I mean, if I resolvate the dry sample, for example in 150uL of solvent and inject 5uL in mass spectrometry, how am I going to use 150uL.

Alternatively, the signal intensity I get from 150uL and 500uL resolvation of analyte is different. How can concentration related to the actual sample before extraction?

Please let me know if the question is unclear?

Thank you again.

As long as you inject the same volume for all samples and calibration standards you can ignore the injection volume. If you make a 1000ul standard with a concentration of 5ug/l concentration and inject 5ul, then you resolvate your unknown sample to 150ul and inject 5ul and obtain a response of 5ug/l, then the concentration of your sample is 5ug/l which is equal to 5ng/ml. Your final sample volume was 0.15ml so you simply take 5ng x 0.15ml = 0.75ng/150ul. This means you had 0.75ng in the sample you evaporated.

If the original sample was 1g before extraction then you have 0.75ng/g of analyte in the sample.

Just work with simple ratios, Concentration1/volume1 = Concentration2/volume2 and solve for the unknown just like making dilutions of standards from concentrates.

Sirius, if you struggle with this sort of calculation (and you won't be alone if you do!) then you might find it helpful to draw out the protocol: I sometimes draw the whole thing as a set of pictures of little vials and tubes, with arrows indicating where I'm adding to a tube, and what I'm taking. I shade in the approximate volumes so the tubes even look sort-of right! I can then work out the quantities or concentrations at each step.

Although this sounds a bit "babyish", I find it a lot easier than trying to work with abstract equations, and less error-prone. It is my standard method of understanding other people's protocols when they turn up asking me how to do the calculations. It's particularly helpful in other people's protocols because, if I haven't done the job myself, I have no graphic image in my mind of what actually happened in the sample prep.

Calculations (in fact the whole or arithmetic) are actually just a model of reality. I find the calculations easier when they're linked very strongly to the reality of what I actually did.

(sorry if this is a bit patronising - I don't mean to be - it's just it's helped me so much I don't want to keep the approach to myself!)

Hi all
I am new to LC/MS/MS its actually my first time, i have read some papers and i want to use IPC-LC/MS to separate metabolites in my sample. But for some reason beyond my control i am unable to obtain or buy TBA due to govt. restrictions. So my question is what other compound can i use since i do not have experience. i read about formic acid and acetic acid but not really sure i can make that call. Just so you know my samples come from Arabidopsis seeds and am looking to quantify ATP---ADP and NADP----NADPH
Please help me.
Thanks

Hey,

If you mean tetrabutylammonium salts with "TBA": it acts as an ion-pairing reagent. Substituting it with formic or acetic acid will not work.

I suggest you check out some more literature for the combination of ion-pairing chromatography and MS before starting to inject stuff. I'm not sure if and which TBA salts are even compatible with MS; I haven't used them.

EDIT: check out this link

Note how they suggest TBA salts for HPLC, but other ones for LCMS

PS next time make a new topic for your question

Thank you very much for your kind assistance, it was very helpful to me. Am sorry for not making a topic i also did't know how to make the topic but i have seen that its quite easy.
I look forward to your assistance if i run into trouble again.
Cheers.