Hello,

do you have the possibility to change the separation (gradient) so the analytes will elute in a region where the ion-suppression is absent or very low?

Or try to furhter improve the sample preparation to get cleaner samples!

Before apply the samples in the LC MSMS system do you use the clean method ?

We usually use CleanUp 2DE kit or a acetone precipitation for clean the samples.

Hi, I agree with Roland, If you can adjust the gradient (this may mean a slightly longer or shorter run time) so your analyte elutes when ion-suppression or enhancement will have the least matrix effect this is the best solution. Good luck.

If you could not change chromatigraphy (column, eluent, pH) or sapmle prep (LLE, SPE,..) you can work with standard addition. Than you have the same matrix in sample and calibrationsample. But its just for a little number of samples. Or try to dilute you sample if your detectionsystem is sensitiv enough.

I suggest to go for clean up step using SPE. If the quantitiation is on the higher side u can use internal standard

I would also recommend spiking the samples with different concentrations of the analyte and then from the obtained calibration curve calculate the proper concentration.

Depending on your MS instrument and amount of analyte, you can try to perform more than one fragmentation (MS^n, n=3,4...) to enhance selectivity. If pure analyte is available, try to find a fragment of high intensity in MS/MS and fragment it once more. This does not work with every MS intrument.

I agree with all the solutions. other than these, you can also consider following points:

1. Use solid phase extraction for better clean up

2. Use different MRM transition

3. Prepare CC's and QC's from single lot of matrix (Which matrix you are using for CC's & QC's preparation?)

How you are determining matrix effect?

Hope this may help you.

Hi

Have you checked the method with low matrix volme?

- Try to run a blank sample and see if your background ions in this spectra are high.

- In that case maybe your column needs tobe cleaned up or changed.

Use standard addition or matrix matched standards

Two most popular options are cited here: use matrix matched controls, or perform clean-up. A third, perhaps unpopular to some, is to consider an alternate method. LC-MSMS is not the hammer for every nail. Regards.

Hello,

if you use standard addition method do not forget to check if there excist an analyte concentration dependance of the ion-suppression effect (is the ion-suppression effect equal at low and high analyte concentration!)

I agree with most propositions given here. Sample cleanup is one option and the other obvious one is standard addition. I am afraid that doing SRM or MSn experiment does not eliminate the matrix effect, as some inferred. Do you observe matrix effects for all your analytes? How do you determine the matrix effect? By post-column infusion?

The standard method to neutralize matrix effect in quantitative LCMS is to use isotopically labelled analytes as internal standard, when available. The quantitation is performed using the ratio analyte/IS (ratio of pek areas). If performed correctly, biasses related to matrix effect is completely and reliably eliminated.

Agree with Alexandra

Matrix-matches standard will be a necessity, even after solid-phase clean-up. Also 3 transitions if possible. But most importantly, if you haven't done so yet, stretch the chromatography (increase slowly the gradient) and use a longer column. Don't rely on vendors demo-chromatogram of "separating 8 compounds in 4 minutes" on a 50mm column...this is OK for theory, but with food extracts, it doesn't work!

If matrix-matched standards is not a possibility, a combined approach of standard addition and internal standards can be employed for better accuracy.

Definitely run a blank control of a matrix sample to ensure that there won't be any background or ion-suppression effects from the matrix itself. Why do you feel you have to have an isotopic internal standard? Is there a compound similar to your target molecule that you might be able to use as an internal standard? Then, you could spike it in from the beginning and definitely be able to recover from matrix effects.

First, check stability of your système (LC-colum and detector) at no injection background situation. Control that there is no baseline fluctuation. Then made external standard injection. Inject 5 times every point, corresponding to an appropriate dilution. If the standard deviation is less than 5% of the average value, then you can do and use as calibration curve with the average value. This support the fact that your matrix effect is not really significant.

At the other hand, if standard deviation is superior to 5% then i think you should have some repeatability problems with your injector. Check if the injector's injection loop is appropriate for the injection volume you use to execute. Perhapps your encreasing matrix effect originate there. ussually MS detection is very good and of course expensive. So I dont think that your probleme will be there. But could you tell us which MS system you have? What kind of compounds (in term of chemical structure or propertie) you are analysing? what is the effective or real composition of your mobile phase? What LC-colum? your flow-rate and working pressure in your pump? is this working pressure constant ? Or it's amount of fluctuation? these questions are pointing to a baseline or system backgroung fluctuation or unstability.

I would rather if i could help you more.

Best regards

DZAHINI

Choose the right solvent for extraction.

Hi again, as Kmamivi Dzahini ask : what compounds are you looking for. I think with the ABI5500 you have a very sensitv MS. So you schould eventually change your mobile phases. By the Water and the Methanol from a tiffrent manufacturer. If you need 100% MeoH, why don't change to acetonitrile. Or is it too much for your early eluting analytes. 14 analytes are not that much we can read but if they are as you wrote polar and non polar - its very tricky to find the right LC conditions. Its possible to check a "AQUA" column? Or if you are working with Phenomenx , check the Synergi PolarRP, Hydro,...

What kind of water is it what you are testing. What ist you bad guy of matrix? Could you let run a Q1 Scan with some diffrent Watersampels, for checking what is in there. If you get some "big" signals, do a compound optimisation to get an MRM. Setup a new method with the same gradient and let run the samples to see when the "matrix" is eluting. If you could find what it is, than you can look if a diffrent SPE would help you.

Dirk

1) The best approach would be a stable isotope dilution assay, but for that you need the related standard for each compound; as matrix effects may affect only small parts of the chromatogram, an internal standard that is not co-eluting does not help anything; the obvious disadvantages are the availability of the standrds and the cost of that approach

2) If you are reluctant to change the LC-MS/MS conditions (use of anther ion source or change to negative polarity could be an option, but probably not all your analytes are amenable to that), try a flatter gradient; that has helped a lot when we have analysed mycotoxins in beer

3) Matrix matching is also an option but in that case I would investigate the difference of extent of the effects between different samples (relative matrix effect)

4) As you are using the AB 5500, a lower enrichment factor of the SPE-clean-up might also help

Of cause there should be many possible reasons for the effect of the matrix. I tried and did not find what kind of mobile phase did you used. I used to add ammonium formate, or acetete to improve the s/n of my lc esi ms analysis. It works well many times. The increase of conductivity of the solution can increase the charge density of the droplet and therefor improve the performance of esi.

Sometimes switching to APCI would help, instead of API (I can't find what ionisation you use). Of course, if your compounds allow that.

We deal in very complex matrices as well, and have found that much of the issue is with the ionization (I assume from your parameters that you are using either ESI or API). The only effective means of correction that we've found is to use known addition. The best solution for us has been to use deuterated analogs; if you're only looking for one or two compounds this is tolerable from a cost standpoint. Otherwise, if we have to move to multicomponent standards we use a 3 point standard addition - sample plus 2 levels of spiking with the compounds. It means you have to make 3 injections per sample, but it's the only way we've found to adequately correct for the matrix.

Despite you said that the matrix effects vary among the individual samples, you can use matrix-matched calibration for the quantification. You still makes an error in quantification, but compared to neat sovent calibration quantification it is not significant.

If there is a matrix problem you can analyze your sample by standard addtion technique

Gea Oliveri Conti

Try to use standard addition method and use LCMsMs system with Q_trap system

Another option could be to use better sample clean-up. In my experience Size Exclusion Chromatography (which you can get a analytical columns as well), can do wonders. So In my ideas using a SEC column performing a heart cut and transfering it to the analitical column could improve your stuff considerably. - I have worked with Phenomenex and polymer laboratories columns. both do a good job. - good luck

I actually agree very much with Kai. However, Since no information on the matrix type is given, it is not clear what strategy for sample clean-up should be preferred. In general SEC/GPC type of chromatographic cleaning will always be the first choice for complex biological samples incl. waste water (grey/ black). In addition isotope labeled ISTDs are always required also for compensation of matrix induced effects.. Please note, by insufficient clean-up you may/will run into matrix induced ion suppression in your MS/MS quantifications,. This will lead to non-linear response problems and will completely destroy your quant . standard calibrations.

You can also use amino or PSA cartridges for purification. But you need first identify your matrix effect. For that you can inject a real sample and in parallel, do an infusion with your sample mix all along the rune. By this way you can "localize" your matrix effect that can pearups not affect all your compounds. Some times, the probleme take places at the first minuts of chromatographic analysis, so you can out this part to the waste to avoid polluting all your system (even if there is no targeted compounds in this part of the chromatogram). Another solution is to inject a lower volume of samples : in some case, it is enough to suppress signal extinction....

And I shw that you use methanol for elution : Have you tried to use Acetonitrile, in some case it is sufficient for extracting the targeted compounds, but with lower matrix extraction associated.

Have you tryed optimize the ions using the analites in matrix? It sometimes helps...do you have blank matrix to construct the calibration curve in matrix? Could you inform the analytes and matrix? Good luck!

You can use the dilution of the sample before injection and then I suggest you follow the fragment ions for the quantification of your analyte.

If that were not enough, then you will need to purify the sample with SPE column

It sounds like you are extracting waste water samples? If the source is from human waste water you are most likely dealing with surfactants and benzalkonium chlorides (BACs) swamping your electrospray interface. These routinely are present at very high concentrations in waste water. Good news is you can remove them effectively post extraction with SPE cleanup. First though I would try and figure out what the interference is by running full scan on your most problematic sample.

A good reference on what to scan for regarding BACs can be found in this journal article doi: 10.1016/j.envpol.2012.01.037 (google the doi, then choose science direct as source) I ran into this exact problem recently, and found BAC C14 was eluting at the exact same RT as one of my targets. The BAC C14 had area counts in the millions, which suppressed my target, luckily my internal standard picked up on it.

If you are using just C18, SPE, you might want to try a different SPE sorbent up front, like Waters Oasis HLB - you can increase %organic (up to 20%) in wash step with these types of sorbents without loss of targets.

Finally - if you are routinely needing to dilute due to interferences, save yourself lots of time and effort by just extracting less. Like 250 mL instead of 1000mL, its only a factor of 4, and if you are having to diltue every sample by 5X ..... well you get what i mean.

While all the clean-up suggestions may be useful they may not necessarily remove the matrix effect enough to give good quantitation. My preference would be isotopic internal std - but it needs to be the target compound of interest. Other than that matrix matched stds can also lead to problems if the matrix effect is not consistent between samples. The cheapest and quickest solution is the analyte addition approach.

Filtration and HLB cartridge use in SPE is a good choice to remove matrix effect. Waters or Sigma HLB is good.