I am using HPLC-MS analysis to quantify MC-LR in presence of Natural Organic Matter (NOM). I am getting a well-fitted curve while preparing the standard sample using deionized water. But, while I am trying to quantify MC-LR in presence of NOM I am not getting good results. It seems the NOM affecting the MC-LR quantification. And I am getting much higher value of MC-LR than expected in presence of NOM. Is there any method that I can employ to improve my results or reduce the impact of NOM in HPLC analysis?
Hello Bayshakhi,
From your description: “I am getting a much higher value of MC-LR than expected in presence of NOM.”
If everything goes right (the instrument is well-maintained and no carryover from the last run), this phenomenon indicates that the ionization efficiency in the NOM-contained sample is quite different from the DI water, which means the standard curve you fitted is not applicable.
Two possible solutions:
1) Using solid-phase extraction (SPE) to eliminate the "matrix effect" before analysis by the MS;
2) Repoltting the standard curve using the NOM-contained water.
It seems you face with matrix effect of NOM. The first choice must be the matrix-matched calibration if you can get MC-LR-free NOM. I mean blank material of NOM. You are performing an external standard calibration approach, therefore it is plausible to get a bit shifted results because MS can be affected by sample composition, unfortunately. SPE is a good approach to decreasing the matrix effects but it may not be the perfect solution. I can recommend using an internal standard as spiking to both sample and calibrants. It can be an analog molecule that has a similar structure to MC-LR or isotopologues (carbon or deuterium) of the MC-LR.
Both answers before a valid. However there is another solution as well.
1) as pointed out before standard addition so You always run your sample and the You run the same sample with standard added.
2) You do Your calibration in NOM containing water
3) You use good internal standards (ideally isotope labelled internal standards)
4) change the ionisation on some machines ESI is quite suceptible to matrix effects, while usually APCI is less prone to give troubles
by the way there is a bunch of literature about this (check for matrix effects in HPLC-MS
Please let us know column, mobile phase, and buffers being used. Sometimes you can adjust these parameters so the MC-LR will elute away from the NOM. I would not recommend adding NOM to calibration standards, as NOM is not consistent from water body to water body. You would need characterize each water body NOM to matrix match for every water body being sampled - and NOM can fluctuate within a water body. Internal standards and maybe even isotope dilution might work - but I would start with the chromatography.
Peter Philbrook The separation was achieved on a Zorbax Extend-C18, 2.1mm×150mm (Agilent technologies, Germany) column. Injection volumes were 5uL. The mobile phase consisted of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B).
Hello - If possible you could run a UV detector in front of Mass Spec to look for NOM - at least then you would be able to determine where it elutes during your run. Then you could try to adjust the gradient to elute the MC-LR away from the interference. I've attached a copy of what we run - I use acetonitrile/methanol/water gradient - this is a little complex as I'm running anatoxin-a and cylindrospermopsin along with the common microcystins. So this gradient is more focused on the front end to separate the more polar target compounds. Using a Waters HSS T3 3.5um 2.1x150 column. You have great flexibility in adjusting gradients with HPLC - you just might be able to get the MC-LR away from the NOM with a different solvent combination - good luck!
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