Chromatography Forum

Hi All,

I hope you can help me with this problem i'm currently having or have any experience with this issue. I'm developing a 37 analyte "dilute and shoot" method on a ThermoFisher Quantis. I'm noticing the Peak areas for only Benzodiazepines are doubling over time. If i Inject 20 injections back to back the RSD of the areas are great but if i let the vial sit for an extended period of time and then re-inject the Benzodiazepine peak areas double but other analyte classes in the method such as opiates/opioids and amphetamines are not.

currently my sample prep is 100uL of standard (made in Surine from Ceriiliant ref material) mixed with 10uL of ITSD (1000ng/mL) and 50uL of a Beta-Glucoronide Enzyme Mix for hydrolysis. After incubation 95uL of MeOH is added and then centrifuged. 25uL of the Supernatant is then diluted to 500uL in H20. The injection volume is 10uL and the mobile phases are A: 0.1% Formic Acid in H2O and B: 0.1% Formic Acid in MeOH. The column is a Phenomnex BiPhenyl.


Im lost over what it could be considering its only Benzos!

Any help would be amazing!

Do you detect Benzo in your 'blank' injections? What is the amount of the organic solvents in your mobile phases?

The blanks are clean there is minimal to no carry over. For the amount of organic solvents I’m not understanding what you’re asking. My organic solvent is just 100% optima MeOH with 0.1% formic acid.

This is interesting! Benzo can degrade (see attached, Chemistry section) but protonation from 0.1% formic acid must occur 1st. I am going to presume this degradant elutes at the same retention time as Benzo AND has a higher detector response factor.

I would try 0.010% formic acid 1st and increment its concentration by 0.010%. In other words you control (and manipulate) the acidity.

Good luck!

So the Area counts are increasing not decreasing though and its for every benzo (Oxazepam, Alprazolam, 7-AminoClonazepam, Clonzaepam, Alpha-Hydroxy-Alprazolam, Nordiazepam and Diazepam) and all those have different retention times.

acmoli01 wrote:
So the Area counts are increasing not decreasing though and its for every benzo (Oxazepam, Alprazolam, 7-AminoClonazepam, Clonzaepam, Alpha-Hydroxy-Alprazolam, Nordiazepam and Diazepam) and all those have different retention times.

Are you using deuterated analogs for internal standards as isotope dilution?

If so you could be swaping a deuterium for a hydrogen, especially if it encounters a pH less than 2 anywhere in the process.

Yes I’m using deuterated ITSDs but they samples themselves are sitting in H20 so there should be a neutral pH up until the point they mix with my mobile phase in the sample loop.

acmoli01 wrote:
Yes I’m using deuterated ITSDs but they samples themselves are sitting in H20 so there should be a neutral pH up until the point they mix with my mobile phase in the sample loop.

That should be ok then, but just watch to be sure you do not have lower ISTD area and higher Target areas, if so then it is something to consider.

Could the problem be with deconjugation? Maybey the extra benzos are present as conjugates that deconjugate in the sample in the autosampler. Tey deconjugate longer and see if yhe area is bigger than admfter cyrrent deconjugation time. Ir try measure conjugates .
( i asume conjugates are glucuronides and sulfates that are comercial avsilable)

I think I’ve found the problem. I remade the mobile phase and it actually lowered the area counts compared to the old mobile phase I had been using. The old mobile phase had been sitting for a week so the formic acid had probably degraded over time in the methanol ( I found an research paper that said 0.1% FA has a half life of 88hrs in Methanol). I then ran the same samples with straight methanol and I observed the same response as I did with the old mobile phase. I’ve always read formic acid helped protonation and signal but I’m seeing the opposite. Has anyone else experienced this? I’m considering just running with methanol and no formic acid.

Adding (formic) acid is no guarantee for a better ESI+ signal. There are a lot of parameters involved, most importantly the signal increase/decrease you get as a function of additive concentration is very analyte-dependant. Find something that works and is within reasonable boundaries for LCMS, and is at a good pH for your separation. If plain MeOH works for your method, I see no reason not to use it.

Thank you!