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- Posts: 2
- Joined: Mon Jun 30, 2014 10:41 pm
Hi all,
I have been having trouble with an LC-ESI-MS/MS method for Estradiol (dansyl-Estradiol). Specifically, I see native Estradiol (506/171) in injections of internal standard only, regardless of the isotope used (d3, 509/171; d5, 511/171, I actually see more d3 than d5; 13C3, 509/171). Samples where IS (diluted in ACN) is dried down, derivatized, and then injected are occasionally fine (no 506/171). Samples that have been through LLE are not, ever. Prep is as follows:
Pipet the following into a 13x100mm glass tube:
500uL sample (BSA or PBS diluted calibrator, or serum/plasma sample)
50uL 500pg/mL IS (diluted in ACN)
500uL H2O (Optima)
2mL MTBE (HPLC Grade)
Cap with a teflon coated screw cap, vortex briefly, centrifuge to separate phases. Pipet organic phase to new glass tube. Dry under N2 at 37C.
To the dried extract, pipet the following:
100uL Ethanol (USP grade)
500uL H2O (Optima)
2mL DCM (HPLC gradE)
Cap with a teflon coated screw cap, vortex briefly, centrifuge to separate phases. Pipet organic phase to new glass tube. Dry under N2 at 37C.
Reconstitute in 25uL 100mM NaHCO3 (pH =10.5), 50uL 1mg/mL Dansyl Chloride (in ACN). Cap, incubate to derivatize (I've used many temp.time combos, all work well- 60C, 5min; 40C, 4min; 30C, 15min). Remove and pipet 25uL Optima H2O to stop reaction. Transfer to HPLC vial and inject.
I always see 506/171 in samples that have been through total prep, and only sometimes in IS only. My FSE has evaluated cross talk and pulled the rails for extensive cleaning. Solvent blanks are beautiful, shortcut IS preps are beautiful. I see this regardless of DP, CE, Spray voltage, gas settings...at this point the only thing that hasn't been explored is the collision cell.
Do you think that I could be dansylating PPG originating from PPE tubes that the dansyl chloride is prepared in? How does this sound: PPG- 251.1, + Dansyl Chloride, 269.7 = Dansyl-PPG = 484.4, and then the sodium adduct would give a parent ion of 506.4, which would produce the dansyl fragment 171, and 156. I'm reaching for straws, but what do you think?
Any insight is greatly appreciated.
Kristin
I have been having trouble with an LC-ESI-MS/MS method for Estradiol (dansyl-Estradiol). Specifically, I see native Estradiol (506/171) in injections of internal standard only, regardless of the isotope used (d3, 509/171; d5, 511/171, I actually see more d3 than d5; 13C3, 509/171). Samples where IS (diluted in ACN) is dried down, derivatized, and then injected are occasionally fine (no 506/171). Samples that have been through LLE are not, ever. Prep is as follows:
Pipet the following into a 13x100mm glass tube:
500uL sample (BSA or PBS diluted calibrator, or serum/plasma sample)
50uL 500pg/mL IS (diluted in ACN)
500uL H2O (Optima)
2mL MTBE (HPLC Grade)
Cap with a teflon coated screw cap, vortex briefly, centrifuge to separate phases. Pipet organic phase to new glass tube. Dry under N2 at 37C.
To the dried extract, pipet the following:
100uL Ethanol (USP grade)
500uL H2O (Optima)
2mL DCM (HPLC gradE)
Cap with a teflon coated screw cap, vortex briefly, centrifuge to separate phases. Pipet organic phase to new glass tube. Dry under N2 at 37C.
Reconstitute in 25uL 100mM NaHCO3 (pH =10.5), 50uL 1mg/mL Dansyl Chloride (in ACN). Cap, incubate to derivatize (I've used many temp.time combos, all work well- 60C, 5min; 40C, 4min; 30C, 15min). Remove and pipet 25uL Optima H2O to stop reaction. Transfer to HPLC vial and inject.
I always see 506/171 in samples that have been through total prep, and only sometimes in IS only. My FSE has evaluated cross talk and pulled the rails for extensive cleaning. Solvent blanks are beautiful, shortcut IS preps are beautiful. I see this regardless of DP, CE, Spray voltage, gas settings...at this point the only thing that hasn't been explored is the collision cell.
Do you think that I could be dansylating PPG originating from PPE tubes that the dansyl chloride is prepared in? How does this sound: PPG- 251.1, + Dansyl Chloride, 269.7 = Dansyl-PPG = 484.4, and then the sodium adduct would give a parent ion of 506.4, which would produce the dansyl fragment 171, and 156. I'm reaching for straws, but what do you think?
Any insight is greatly appreciated.
Kristin
