I have a tough nut to crack. I spiked a mixed standard (Amitrole, ETU, PTU, Cyromazine, Mepiquat, Chlormequat, and Daminozide into 10 g apple and stored them in a freezer over the weekend and extract with different solvent such as
1) 1% formic in methanol
2) 1% ammonium hydroxide in methanol
3) 1% ammonium hydroxide in acetonitrile
4) 1% sodium hydroxide in methanol
5) DMSO
6) Dimethyl formamide
7) Dilute acetic with EDTA (glyphosate solvent)
8) 1:1 methanol:0.1 N HCl room and 80 C 15 min (paraquat solvent)
9) 1g sodium acetate + 10 mL methanol (Richard Krause JAOAC 1989 method)
10) Methylene chloride (Simon Hird) method
The issue is I got all but ETU and PTU. These two analytes are very hard to extract. If I spike and vortex and wait for 5 min, I got good recovery just solvent 1. But if I let it sit for 1 hour at room temperature, no ETU or PTU recovery. There are a lot of papers for ETU/PUT in fruit and vegetable out there and they had good extraction yield; however, I suspect that they are all freshly spike or did not indicate that they store before extraction.
Thanks,
Narong
I found that the poor recovery of PTU and ETU only occurs in apple. When I spiked in sugar snap and let it sit for 1 hour, extract with QuPPe solvent (1% formic/MeOH) then it is ok. Something in apple may bind ETU/PTU strongly. Get some out with acetone + 1% NH4OH.
I spiked the standard mix into grape, bean, and snap peas and let them stand for 30 min and extract with 1% formic in methanol (QuePPE), shake 10 min, centrifuge and filter. The recovery for all including ETU and PTU are good. Next, is spike and store in a freezer and will extract them 1 day and 3 days after storage. Apparently, apple will absorb ETU and PTU just after 5 min that any solvent cannot take them out.
ETU is prone to oxidative degradation during extraction and proposed the use of cysteine hydrochloride as a protectant. PTU also might have the same problem.
Thanks, I thought about it and use DTT Dithiothreitol (antioxidant from my folic acid method) into methanol but still not good. I noticed that poor recovery will occur in fruits sample including (apple, pears, another grape sample) but good recovery for vegetable including (sugar snap, beans, spinach). I will try more matrix e.g. lettuce, kale, tomato, broccoli and more today. If it is the oxidation, I should have low recovery for vegetable as well. Apple should have vitamin c (ascorbic, good antioxidant) that will protect oxidation. Right now, I suspect high sugar content in the samples.
I found the answer, ETU will degrade rapidly to EU once it is spiked on the matrix, but not in extracting solvent containing matrix. To fix it, I need to put an antioxidant into the sample, mix real well and then spike ETU/PTU.
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