OPNA metabolites

[montucky1]

using Agilent 6430 and 1200 series HPLC to quantify OPNA metabolites in urine. get peaks and marginal separation, but unable to get good linearity. vx is the worst. is it sample prep? instrument settings.....so many variables.
anyone been there done that? need help.

[Bigbear]

I would bet on sample prep. We do it on an Agilent system as well.
What we found that really helped is to first add your internal standard to 3 ml ACN and vortex. Add the 1 ml of sample vortex, then add 3 more mls. of ACN and vortex. Take to dryness @ 70C. ( vortex samples at 4 min intervals while drying 2-3 times).
We use a Zephyr extractor so at this point we add 900ul 5%H20/ACN ( to the dried sample) and vortex, transfer to a well sample plate. We repeat this step once more so we have about 1.7 ml or so in the plate. We then extract 1 ml of the sample using Strata -SI 96 well plate. We then dry the extracts @ 70c and reconstitute with 100 ul of the 5% H2O, cover and using a plate shaker, agitate for 20 sec or so.

I got lucky and passed the most recent PT with only 1 miss ( a false positive)
it met all out reporting criteria!!!!!!!

[montucky1]

thanks!!
we don't have a zephyr...i'm doing the same steps...went to the onsite training, but can't quant VX or GF
lowered the dwell time to get more samples...starting to think I'm not getting enough ionization. are you using jetspray? if not what is your neb. press?
Thank you so much for helping.

still need to validate

[Bigbear]

We don't have jet spray. Our neb pressure is 60psi, and we use a dwell of 1.36 for GF and 1.2 for all the rest.
Are you sure you're not washing the compounds off prior to elution? We wash with 8% water in ACN.

If you don't mind me asking, where are you located? PM me if you still need help.

[montucky1]

i have discovered the bulk of the issue.... a box was checked deep down in the darkest recesses of Mass Hunter that only an engineer could find... it wasn't allowing quant to "see" the peaks. (thank goodness for webex sessions)
Great cal curve now as long as i don't include the last 2 stds. hoping to solve that problem sometime this month...
i am in montana...Helena.

thank you for all your help on all my issues