By Anonymous on Sunday, February 29, 2004 - 10:57 am:
Dear all,
I am really confused by a calibration curve from a LC-MS/MS instrument. The curve is from 1 ng/ml to 100 ng/ml. The standards were prepared in water/acetonitrile soluiton by spiking from stock solution, not by serial dilution. There is no other matrix. I got decent signal from 100 ng/mL, however, at 50 ng/mL level, the signal is only 1/4 of the signal from 100 ng/mL, then at 10 ng/mL, the signal is only 1/50 of the signal of 100 ng/mL, at the end, the 1 ng/ml solution generated a signal that is only 1/700 of the signal from 100 ng/mL. The final calibration curve is not linear at all, but a flipped quadratic one. As concentrations become higher, the signals increase dramatically, much higher than what you would expect from a linear curve...I repeated three times, got similar curve every time.....
If you dilute a solution from 100 ng/mL to 10 ng/mL, would you expect to see a signal only decreases to 10% of the original one?
Anyone has seen this before? Any suggestion? Thanks a lot!
By MG on Sunday, February 29, 2004 - 06:38 pm:
Yeah, I saw that on one instrument, and then only with certain compounds, but not quite as dramatic as what you're seeing. The manufacturer said I must be using weird conditions. I sent them the conditions, which were normal, and then they said that there must be a contaminated lens somewhere. I moved the analysis to a different instrument. Problem solved.
What's your ionization mode? What kind of compound is your analyte? Is this behavior compound specific? Is it instrument specific? (if you have other instruments). Do you see different behavior if you run your calibration curve in reverse?
By Anonymous on Sunday, February 29, 2004 - 07:34 pm:
Hi MG,
Thanks for the message. I was using ESI-MS, the compound is a polar one, with multiple charges on it. This behavior does appear to be compound specific, but I just don't know why on earth you would expect to see something like this with any compound. I did run the curve in reverse, similar strange results.
Wish I have another instrument I can test, but I don't....
By Anonymous on Monday, March 1, 2004 - 04:15 am:
Probable causes: carryover, absorptive loss.
By MG on Monday, March 1, 2004 - 06:30 am:
Running a blanks in between injections will rule out carryover. Running the curve in reverse should rule out adsorption, I think.
When I had the problem, it was with a polar compound in APCI(+), no multiple charging.
I don't have experience quantitating compounds that form multiple charge states. Perhaps the charge state distribution is concentration dependent?