LC-MS/MS seeing a drug peak in my blank

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

9 posts Page 1 of 1
For a little background I'm currently using a waters TQ-XS instrument with a C18 BEH 2.1 x 100 column for analysis of my drug with an isocratic mobile phase of 73% organic and 27% aqueous
I have run into a problem where I inject a blank (my mobile phase composition) and the first injection will run normally, but when I repeat an injection from the same vial immediately after I will get a peak for my drug that shows up. I can't figure out where the drug peak is coming from when the first injection runs fine.
Any advice?
Thanks!
The problem is this molecule is partially retained and elutes into the next chromatogram, even if it is a blank. Your column is contaminated including the rest of the HPLC. You will have to reverse the column and wash with at least 10x column volumes of 100% ACN or MeOH. Also make multiple injections (100 uL) of the same solvent into the HPLC during the column washing.
Finally you will have to revise your method, since your molecule is being partially retained you will have to (incrementally, 1%) 'drive' 100% off the column by increasing the amount of organic solvent or use a solvent that has a higher strength (like IPA).
BEH columns used for UPLC have a higher hydrophobicity (thus are more retentive) than regular C18 columns so HPLC methods are not directly transferable without adjustment!
pnlane wrote:
For a little background I'm currently using a waters TQ-XS instrument with a C18 BEH 2.1 x 100 column for analysis of my drug with an isocratic mobile phase of 73% organic and 27% aqueous
I have run into a problem where I inject a blank (my mobile phase composition) and the first injection will run normally, but when I repeat an injection from the same vial immediately after I will get a peak for my drug that shows up. I can't figure out where the drug peak is coming from when the first injection runs fine.
Any advice?
Thanks!


Does it happen only after the first injection? Such as, if you place a fresh vial in the autosampler and make injection and it is clean then all injections after that show the peak, then place a new fresh vial in the autosampler, does that one appear clean on the first injection and contaminated on following injections?

If that is what happens then suspect a siloxane from the septa contaminating your vial that just happens to have similar properties to the analyte of interest.
The past is there to guide us into the future, not to dwell in.
Does the peak in the blank show up at the retention time of the drug, earlier, or randomly?
Hplc chemist: I don’t believe it is being retained on my column I have washed it with a high organic for an extended period of time and only injected blank samples in which every time I will not see anything in the first blank injection and then when I immediately inject that blank again without injecting a sample with my molecule in between I will see a peak matching the retention time of my molecule

James_Ball: that is exactly what happens I will inject a blank see no peak, inject it again immediately see a peak, change to a new vial not see a peak inject the new vial again and immediately see a peak. I have changed vial and vial caps and still seems to happen

Rndirk: yes it shows up at the same retention time as my molecule no matter what I do I have tried changing columns, changing column chemistry, changing Pipettes, moving to a different lab space for sample prep, using unopened vial/caps, changing the solvents in the system, changing ph no matter what I do it always shows up on the second injection
Then the most logical explanation is that, as James said, your analyte is present in the septum of the vial. I find it hard to believe that "a drug" is present in the septum. If it is an interference, it has to be a damn good one to both have the same retention time, and the same MRMs (assuming you're detecting in MRM). If it's indeed MRM, does it have the same ion ratio as your drug?

Another question: is the injection flow through or fixed loop?

As an easy test I suggest to try the same experiment with a vial capped with only aluminum foil.
Rndirk wrote:
Then the most logical explanation is that, as James said, your analyte is present in the septum of the vial. I find it hard to believe that "a drug" is present in the septum. If it is an interference, it has to be a damn good one to both have the same retention time, and the same MRMs (assuming you're detecting in MRM). If it's indeed MRM, does it have the same ion ratio as your drug?

Another question: is the injection flow through or fixed loop?

As an easy test I suggest to try the same experiment with a vial capped with only aluminum foil.


Or a run with uncapped vial if the solvent is not highly volatile.

I had this same problem when analyzing for Vitamin D. When I extracted my samples in plastic 50ml centrifuge vials I had a hit at the exact same time as Vitamin D with only a very slight difference in the primary and secondary ratio when using MRM. When using glass vials to do the extraction the samples were completely blank. (was doing a study on powdered plumbs to check for Vitamin D). Either Vitamin D is being added to polyproplyene centrifuge vials or there is a phthalate that gives MRM masses that mimics it quite well. I am sure if we had been using tighter mass windows it probably would have not been as bad, but for sensitivity with such analysis most use a unit mass resolution to accommodate any mass drift during the run.

If it is the vial septa, especially if using silicon rubber, you may want to try the caps that have just the thin polypropylene membrane. They won't reseal completely after injection but will still prevent most evaporation for runs lasting a few hours if that vial has to be injected more than once. Natural rubber septa is also an alternative. You just have to see what other septa material is available and find one that works.
The past is there to guide us into the future, not to dwell in.
I can try to repeat the experiment using only glass and leaving the vial uncapped but I have tried using different caps already and it didn’t seem to make a difference
It is also a flow through needle system
9 posts Page 1 of 1

Who is online

In total there are 7 users online :: 1 registered, 0 hidden and 6 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: Baidu [Spider] and 6 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry