Generally for sample preparation is it recommended to use solvent which matches initial gradient of the mobile phase? For example, if the initial gradient of the mobile phase is 5% acetonitrile and 95% 10 mM ammonium acetate, then is it advised to use similar composition to prepare samples for optimization of different compound depend parameters? How presence of formic acid or acetate ion affect the initial MS parameter optimization? Also, is it recommended to match initial mobile phase gradient to begin with low organic to avoid early eltuion of analytes? As a general practice, i always use solvent matching to the initial solvent composition of the final LC MS method. And for MS parameter optimization I use either water with formic acid or acetate ion. I will really appreciate your expert opinion and any references you might want to refer.
Thank you.
Hi Navid,
your question cannot be answered with a single sentence. I will therefore try to give you practical tips based on the procedures in our laboratory. 1. In principle, we work with water and acetonitrile as eluents (the explanation why would now go beyond the scope here). 2. The analytical systems start with initial conditions of 95/5 (water/acetonitrile). Additive is usually 0.1% TFA. 3. Column temperature is 60°C. 4. The delivered compounds are dissolved in solvents which are also miscible with water. These range from methanol, ethanol, DMSO, DMF, tetrahydrofuran, dioxane to acetonitrile in pure form or mixtures. 5. The injection volume is 2 µL. 6. The RP columns used have the dimension 2.1x50mm with particle sizes of 1.7 µm to 3.5 µm. 7. The samples coming into our laboratory for purification are aliquoted (5 µL sample is diluted with 195 µL water/acetonitrile (50/50). This diluted solution (concentration approx. 10mM) is used for the analytical LC/MS. After several years of experience and several 100000 analyses I can tell you: It works really well. There are two things you need to be aware of: Your solvents must be miscible with water and your sample should not precipitate. Below I have added some practical links concerning solvents/additives and LC/MS.
Regards
Markus
http://www.waters.com/waters/de_CH/Solvents-and-Caveats-for-LC-MS/nav.htm?cid=10091173&locale=de_CH
https://www.shimadzu.com/an/lcms/support/lib/lctalk/60/60lab.html
https://www.researchgate.net/post/Which_solvent_is_recommended_for_use_other_than_DMSO_in_ESI-MS
https://onlinelibrary.wiley.com/doi/pdf/10.1002/0471736589.app2
https://www.acros.com/myBrochure/LCMS_wht%20paper_FINAL.pdf
https://www.sigmaaldrich.com/technical-documents/articles/analytical/bioanalytical/lcms-spice-cannabinoids.html
https://www.chromatographytoday.com/article/lc-ms/48/merck-kgaa/pyes-increasing-lc-msnbspsensitivity-can-be-that-simplep/2308
http://www.rsc.org/images/AMC%20LCMS%20Guide_tcm18-240030.pdf
It is always recommended that you reconstitute samples in a solution similar to the initial mobile phase conditions used. As Rosa stated, it helps to avoid the occurrence of non-Gaussian shaped, early eluting peaks. This can result in a loss of sensitivity of the analytes affected, particularly those at low concentrations or when dealing with trace analysis
Please allow me to add a different angle to the discussion... You asked about sample prep, and the solvents to use... Should they or should they not match the starting conditions...
Shocking answer: It doesn't matter.
Why?
Because it will depend on the sample preparation you do. Usually sample prep is there to remove interfering components. So do that first. Get rid of the interference, foremost.
Once that is done, think about how to get the prepared/clarified sample in a composition that it fits to your analytical determination. (important for the reasons mentioned in previous contributions of our colleagues).
Matching the starting conditions of the analytical gradient usually is a wise decision, no matter what detector (yes, MS is a detector for me as chromatographer, no matter how complex they might be). I find RPLC more forgiving than HILIC, and even less impact I see in IC. This doesn't mean there are none...
Matching the starting conditions of your analytical run can mean a lot. Dilute with organic solvent, dilute with water, dilute with a buffer (e.g. including formic etc), evaporate the solvent and reconstitute with the eluent starting composition of your gradient. I am sure there are plenty more. The decision will depend on where you start after having removed the interfering components.
From my point: Take it step by step. Sample prep first, then match the starting conditions of the analytical run choosing the easiest approach.
I hope I could provide a little "pragmatic" insight in this topic. Crossing my fingers and good luck.
Dear Navid J. Ayon,
I appreciated the answers provided by Markus Christ and Detlef Jensen. In addition, you have to select proper gradient elution system for better separation. Also, solvent composition at the end of solvent program should be good enough to wash out the impurities coming from the samples.
regards
Piyal
Dear All,
Thank you everyone for very insightful discussion. I understand there can not be any single rule of thumb when it comes to sample preparation or solvent selection or gradient selection. It has to be tailored according to the chemical nature of the analytes. My understanding is that if the initial gradient of the method is lets say for example 5% organic and if the sample is made with 50% organic, then it would be a problem if the sample injection volume is large. If the injection volume is lets say 5uL it may be alright. But exceeding 10uL injection volume with a high difference between sample organic percentage vs initial organic % of the gradient can cause early elution or lower sensitivity. Pleses correct me if I am wrong. In our lab we generally do a 20uL injection which might be a problem if there is a big difference between the organic percentage of sample vs gradient. Now for standard commercial entities it's not a problem as we can make higher concentrated stock and inject a small volume to get same pmols injected from a small injection volume. But for some biological samples, where the analyte's presence is low even after concentrating the dried extract and sensitivity is less, it might warrant to use a bigger injection volume. And in that scenario, having the similar organic % in the sample might help to get good sensitivity and elution. Please correct me if I am wrong.
Once again thank you so much for your time and kind responses.
Dr. Markus Christ , thank you so much for the references and sharing your protocol. Dr. Rosa Busquets , thank you so much for your insightful discussion on injection volume and non-Gaussian peak shape. Dr. Detlef Jensen , thank you so much for your advises regarding sample clean up. In one of my other discussion thread, I got some references which are now 1st line of choices for removing interfering entities from biological sample which I cant remember right of hand. But thanks a lot for taking time out to provide your valuable advises.
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