Difficulty with Castor Oil assay

[Larry]

Hello all,

I have a method developed for castor oil which is killing my column after 100 injections. I'm not sure if it is the extraction method or just the castor oil itself.

Column: Resolve C18 5um, 3.9x300mm
Mobile Phase: 83:17 ACN:H2O, isocratic

Extraction
1. 2.5 mL of 40:10:1 isopropanol:heptane:2M phosphoric acid is added to 500uL of spiked plasma, vortex, sonicate, incubate
2. 1mL of heptane and 1.5mL of water is added, vortex, sonicate, centrifuge
3. 1.4 mL of organic layer is removed to clean test tube and dried down by nitrogen stream.
4. 50uL of 15mg/mL 2-bromoacetophenone and 50 mL of 25mg/mL triethylamine are added, vortexed, capped and heated at 100 C, cooled
5. 75uL of 10mg/mL of acetic acid solution is added, vortexed, capped and heated, cooled.
6. sample is dried down by nitrogen stream.
7 reconstituted in 200uL of mobile phase, 100uL is injected, and detected by UV

I have linear curves but I've been through two columns in under 300 injections. Can anyone possibly identify the source of my problem? Thanks.

[Mark Tracy]

What is the symptom of failure? Loss of resolution/symmetry/efficiency? Loss of selectivity?

I would buy a) a pre-column filter and b) a good stock of guard cartridges. Change them at the first sign of trouble.

[Larry]

What is the symptom of failure? Loss of resolution/symmetry/efficiency? Loss of selectivity?

I would buy a) a pre-column filter and b) a good stock of guard cartridges. Change them at the first sign of trouble.

sorry i forgot to mention i am using a pre-column. it was the first thing i troubleshooted. it is only when i change column that issue is resolved. the peak is missing all together.

[Mark Tracy]

I suspect that your column is fouled with strongly retained co-extractives. If you are using a reversed-phase column, lipids can be retained for a long time, and will only be removed by very strong solvents (acetone, methylene chloride, heptane, etc).

[WK]

Larry,
In the long term would your analysis be transferable to GC?
Have you access to GC? Castor oil is detectable nicely when methylated.
WK

[Larry]

Larry,
In the long term would your analysis be transferable to GC?
Have you access to GC? Castor oil is detectable nicely when methylated.
WK

no, only LC