1. Method development for metabolites of CWA-related compounds in mussel and fish samples
Hanna Niemikoski
VERIFIN/ Hanna Niemikoski

2. Aim of the study
Method development for metabolites of CWAs from fish and mussel samples:
Synthesis of reference chemicals for metabolites of arsenic containing CWA’s and sulfur mustard
Synthesis of isotope labelled internal standards
Sample preparation for mussel tissue, cod tissues and bile/urine
Optimization of LC-MS conditions for target analytes
Sample analysis
VERIFIN/ Hanna Niemikoski

3. Timeline
VERIFIN/ Hanna Niemikoski

4. Possible metabolites of arsenic related CWA’s in fish and mussel
Mussels and fish have GST activity [1,2] → glutathione (GSH) conjugates
GSH conjugates are hydrolyzed further → cysteine and N-acetylcysteine conjugates are formed (more stable species)
Arsenic binds to cysteine residues of proteins → arsenic-protein complex can be isolated and after enzymatic digestion arsenic-peptide complex is possible to analyze
VERIFIN/ Hanna Niemikoski

5. An example of in vitro experiment with plasma and CLARK I
Plasma was exposed to CLARK I
After isolation of proteins and enzymatic digestion CLARK-cysteine adduct was detected
Mobile phase: 0,1 % FA in H2O and 0,1 % FA in MeOH
Stationary phase: C18
Ionisation: ESI+
MRM transitions:
m/z 350 → m/z 278
m/z 350 →m/z 229
VERIFIN/ Hanna Niemikoski

6. Hypothesis: Possible metabolic pathway for glutathione conjugation of CLARK I
VERIFIN/ Hanna Niemikoski

7. Sample pretreatment method
Target metabolites of DA and DM for screening of mussel and fish samples
Metabolite
Category
Reference chemical
Matrix
Sample pretreatment method
LC-MS/MS method
As-GSH
Possible metabolite of CLARK I and Adamsite x
Fish (bile, urine, liver)
Mussel (DG)
 Under development
As-Cys
As-NAC*
Fish (bile, urine,liver)
As-protein complex
Protein adduct of CLARK I
X**
Fish and mussel tissues
Under development
*N-acetylcysteine
** Analyzed as cysteine conjugate of CLARK I after enzymatic digestion
VERIFIN/ Hanna Niemikoski

8. Sulfur mustard metabolism
Sulfur mustard forms protein and DNA adducts and glutathione conjugates [3,4]
VERIFIN/ Hanna Niemikoski

9. Sample pretreatment method
Target metabolites of sulfur mustard for screening of mussel and fish samples
Metabolite
Category
Reference chemical
Matrix
Sample pretreatment method
LC-MS/MS method
S-HETE-CysProPhe
 Albumin adduct x
 Fish blood
S-HETE-Val
 Haemoglobin adduct
O6-HETEG
DNA adduct
Fish blood, urine
S-HETE-GSH
glutathione metabolite
FIsh urine/bile
SBMSE
 β-lyase metabolite
 Fish urine
MSMTESE
SBMTE
VERIFIN/ Hanna Niemikoski

10. DNA adduct of sulfur mustard LC-MS/MS spectra
Mobile phase: 0,1 % FA in H2O and 0,1 % FA in MeOH Stationary phase: C18 Ionisation: ESI+ Collision energy: 10 V
VERIFIN/ Hanna Niemikoski

11. Possible challenges
Study of metabolism is essential, there is no information which metabolites are formed in fish and mussel
Complex sample matrix
Stability of metabolites
GSH derivatized adducts
Protein and DNA adducts
VERIFIN/ Hanna Niemikoski

12. References
1. Hellou, J. Environ. Sci. Pollut. Res. Int. 2012, 19, Blanchette, B.; Feng, X.; Singh, B. R. Marine biotechnology. 2007, 9, Xu, H.; Nie, Z.; Zhang, Y.; Li, C.; Yue, L.; Yang, W.; Chen, J.; Dong, Y.; Liu, Q.; Lin, Y. Toxicology Reports. 2014, 1, Halme, M.; Pesonen, M.; Hakala, U.; Pasanen, M.; Vähäkangas, K.; Vanninen, P. Rapid Communications in Mass Spectrometry. 2015, 29,
VERIFIN/ Hanna Niemikoski

13. Thank you for your attention!
VERIFIN/ Hanna Niemikoski