I try to analyzed Thiram as an intact molecule by LC/MS in the vegetable sample. I can get good peak shape of Thiram standard in acetonitrile using a mixed-mode column. The calibration is good from 10 - 1000 ng/mL with 1 uL injection. I made a blank extract of sugar snap using QuEChERS extraction (10 g sample + 10 mL acetonitrile + 0.5 g ascorbic acid). I used ascorbic an antioxidant. After salt out, I spiked 200 ng/mL in the extract to compare with standard in acetonitrile at the same concentration, just to check the matrix suppression. I don't see the peak in blank sample after 10 min of spiking. The compound degraded rapidly under this condition. A reference I got recommend to use sulfite buffer pH 12 to extract but Thiram will split in two and they will analyze the anion which is not specific. I need to find the way to preserve the compound spiking on the sample and during extraction. Most method they spike on the surface (no chopping or blending for the fear of enzyme in plant will degrade the compound) and shake sample with solvent in plastic bag. Any suggest is welcome.
Thanks,
Narong
Try using their extraction and clean up procedure.
https://www.researchgate.net/publication/237146847_Dispersive_liquid-liquid_microextraction_of_thiram_followed_by_microvolume_UV-vis_spectrophotometric_determination
Article Dispersive liquid-liquid microextraction of thiram followed ...
There is a good research paper on Thiram stability issue when spike Thiram into fruit sample. Enzyme from cell will quickly degrade Thiram in less than 1 hour. Most people spike on the surface of uncut sample and shake with solvent. I need to find a way to deactivate the enzyme.
Hello Narong!
I notice that Thiram is highly soluble in ether. Perhaps this is a case where you perform a more traditional extraction into ether or pet ether and then exchange the solvent back into acetonitrile for HPLC analysis? It is probable that the enzyme is not soluble in non-polar solvent.
Latest, by extracting sample with ethanol, the stability in solvent of Thiram is good. Recovery is better than acetonitrile. The recovery is as near as 100 % of standard Thiram in the same matrix (apples, pears, green beans). The trick is spiking method. Many papers suggest to spike in solvent, other extract immediately to avoid enzymatic degradation. If spike in sample matrix and let it sit, Thiram will degrade rapidly. I used dry ice to chop the sample in a Robocoup to get homogeneous texture and minmize enzyme activity. Some papers spiked Thiram on the surface of the sample and make the calculation of ng/g sample a bit tricky. If I spike in a big produce (not minced) such as apple, I will have different ng/g than small produce such as black berry. The matrix suppression can be fixed by internal standard d12 Thiram, no problem. Any suggestion on this?
@ Susana: Thanks for the reply. Yes I tried adding 0.5 g EDTA into 5 g sample with 20 mL solvent (ethanol or acetonitrile) and did not find Thiram in the extract. The reason may be because I shake on a Geno grinder (instead of sonication). Acid definitely will degrade Thiram. At high pH (10 or 12) with sodium sulfite will change the molecule to anion and no longer the intact molecule. I ground the sample with dried ice and keep in the freezer. So far the method I have is 5 g sample with 20 mL ethanol and spike Thiram in solvent. I use sonication for 30 min instead of shaking on a Geno grinder (to reduce contact of oxygen in the air). I will try adding EDTA with sonication in ethanol later. I hope that ethanol will denature the protein (80% ethanol final concentration). The recovery of Thiram with matrix matched standard is 70%. With acetonitrile, I can get about 60%, but with ACN, I may be able to separate water from ACN using salting out (QuEChSERS). The calculation can be tricky and unconventional if I spike on the outer layer of produce and account for all the weight. I attached one of the paper I have. It looks like I have to do the best I can. Today, I will be looking for other analytes in the group to see if I can see it on my mixed-mode column.
yes, Susana, I have your paper with Prof Hernandez as well. I like your paper because you determine the intact molecule not the anion under alkaline pH.
@Susana: Any recommendation of how to make a clear standard solution at 1 mg/mL for Ziram, Zineb, Maneb, and Propineb (cloudy)? I use acetonitrile for Ziram and the rest is Na2HCO3 pH 12 with NaOH but not in solution. I try to infuse the solution to get the mass spectrum and evaluate the retention time. No problem with Dazomet (ACN) and Ferbam (ACN).
Thanks,
Narong
I saw a good poster and paper mentioned the role of reducing agent and the presence of metal ions that may degrade Thiram to anion and reduce the recovery. EDTA is used to fix this issue. This is a good idea.
I found a paper by a Japanese chemists running Ziram by APCI using a gel permeation column and they used chloroform to dissolve Ziram standard. So, I used dichloromethane and got clear solution of 1 mg/mL. Dilute with acetonitrile and infused ESI and got mass spectra and MRM of 305/88. The sensitivity is not that great and was retained (on not well retained ) at 1.2 min similar to Thiram and Dazomet. My column is 100 x 3 and at 0.5 mL/min, the t0 (non-retained) should be approximately 0.5 min.
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