Speciation analysis of lipid-soluble arsenic compounds (arsenolipids) in marine oils new compounds and new challenges Jens J. Sloth, Veronika Sele and Heidi Amlund
Arsenolipids? - only for geeks or nerds? trendy technology mathematics superman scientist biochemistry autistic
How does arsenic speciation fit here? ias in rice ias in seafood Arsenolipids?
Arsenic species in marine samples Total Arsenic Lipid-soluble arsenicals Water-soluble arsenicals? Arsenolipids Organic arsenicals Inorganic arsenicals Arsenite Arsenate CO O- R Arsenobetaine (AB) Arsenocholine (AC) Dimethylarsinoriboside Trimethylarsinoriboside Methylarsonate (MA) Dimethylarsinate (DMA) Trimethylarsine oxide (TMAO) Tetramethylarsonium ion (TETRA)
Analytical progress in analysis of arsenolipids 196-7s: First reports on total arsenic in marine oils. Gulbrand Lunde, Doctoral thesis, Oslo, 1974
Total arsenic in marine oils Marine oils: up to 5 mg As/kg oil. Fish oils:.2 16 mg As/kg oil. Terrestrial oils <.1 mg As/kg oil. Arsenolipid review publication: V. Sele, National J.J. Sloth, Food A-K. Institute, Lundebye, Technical E.H. Larsen, University M.H.G. of Denmark Berntssen and H. Amlund, Arsenolipids in marine oils and fats: a review of occurrence, chemistry and future research needs, Food Chem, 212, 133, 618-63.
1988: Identification of first arsenolipid (Morita & Shibata, 1988) Chromatographic extraction Saponification Photo: http://soer.justice.tas.gov.au/29/i mage/866/index.php GC-MS H-NMR ICP-AES 1988-24: Analysis of hydrolysis-products Phosphatidylarsenocholine Dimethylated arsenolipid(s)
Analysis of intact arsenolipids by LC-ICPMS 8 Abundance Abundance 38 7 36 65 34 32 6 3 55 28 5 26 24 45 22 4 2 35 18 16 3 14 25 12 2 1 15 8 6 1 4 2 5 Time--> TIC: 13SMPL.D (+,-) TIC: 5SMPL.D (+,-) 2. 4. 6. 8. 1. 12. 14. 16. 18. Industrial fish oil 12 Total As = 1.8 1.1 mg/kg NP-HPLC-ICPMS Mobile phase: acetone/hexane/hac/tea 4 main arsenolipids + some minor No info on chemical structure Special instrument settings needed - Narrow torch, O 2 addition, cooled spray-chamber, solvent-tolerable spareparts, low solvent flow, Pt cones Sloth and Amlund, 27.
Types of arsenolipid compounds Arsenosugar-phospholipids (As-sugPLs) R R R= Arsenic-containing fatty acids (AsFAs) Arsenic-containing hydrocarbons (AsHCs) Trimethylated arsenic-containing fatty alcohols (TMAsFOH) Taleshi et National al., 28; Food Rumpler Institute, et al., Technical 28; Morita University & Shibata, of 1988; Denmark Garcia-Salgado et al., 212; Amayo et al., 211+213, Sele et al., 212+13+14
The arsenolipid family: Arsenic Fatty Acids (AsFA) Arsenic Hydrocarbons AsFA264 C1 H22 As O3 (AsHC) Arsenosugar phosphorlipids (AsPL) AsFA276 C11 H22 As O3 AsHC34 C15 H34 As O AsPL93 C43 H85 As O14 P AsFA32 C13 H24 As O3 AsHC33 C17 H36 As O AsPL944 C44 H87 As O14 P AsFA34 C13 H26 As O3 AsHC332 C17 H38 As O AsPL954 C45 H85 As O14 P AsFA316 C14 H26 As O3 AsHC346 C18 H4 As O AsPL956 C45 H87 As O14 P AsFA328 C15 H26 As O3 AsHC358 C19 H4 As O AsPL958 C45 H89 As O14 P AsFA334 C15 H32 As O3 AsHC36 C19 H42 As O AsPL972 C46 H91 As O14 P AsFA348 C16 H34 As O3 AsHC374 C2 H44 As O AsPL98 C47 H87 As O14 P AsFA356 C17 H3 As O3 AsHC388 C21 H46 As O AsPL982 C47 H89 As O14 P AsFA362 C17 H36 As O3 AsHC42 C22 H48 As O AsPL984 C47 H91 As O14 P AsFA374 C18 H36 As O3 AsHC34 C15 H34 As O AsPL986 C47 H93 As O14 P AsFA376 C18 H38 As O3 AsHC33 C17 H36 As O AsPL1 C48 H95 As O14 P AsFA386 C19 H36 As O3 AsHC332 C17 H38 As O AsPL112 C49 H95 As O14 P AsFA388 C19 H38 As O3 AsHC346 C18 H4 As O AsPL114 C49 H97 As O14 P AsFA39 C19 H4 As O3 AsHC358 C19 H4 As O AsPL128 C5 H99 As O14 P AsFA44 C2 H42 As O3 AsHC36 C19 H42 As O AsPL138 C51 H97 As O14 P AsFA418 C21 H44 As O3 AsHC374 C2 H44 As O AsPL142 C51 H11 As O14 P AsFA422 C22 H36 As O3 AsHC388 C21 H46 As O AsPL17 C53 H15 As O14 P AsFA424 C22 H38 As O3 AsHC42 C22 H48 As O AsFA436 C23 H38 As O3 AsHC44 C23 H38 As O Trimethylated arsenoyl AsFA446 C23 H48 As O3 fatty alcohols (TMAsFOH) AsFA448 C24 H38 As O3 AsAH374 C2 H44 As O AsFA474 C25 H52 As O3 AsAH418 C24 H4 As O AsFA52 C27 H56 As O3 Ásta Petursdottir, Phd thesis, Univ of Aberdeen, 214
Analytical challenges in speciation analysis of arsenolipids Instrumentation Sample matrix Extraction Organic solvents Arsenolipids Identification Unknown species Quantification Certified Reference material Available standards
Extraction of arsenolipids Total arsenic Organic solvent Aqueous solvent Lipid-soluble arsenic Water-soluble arsenic Photo: NIFES Methanol/water Hexane Polar arsenolipids Non-polar arsenolipids Concentrated in arsenic 95-99% of the lipid matrix Rumpler et al,. 28; Taleshi et al., 28; Schmeisser et al., 26; Taleshi et al., 21; Sele et al., 212; Sele et al., 213
Analysis of the AsHCs by GC-ICP-MS Signal (cps) 8 4 A B Anchovy Signal (cps) 4 2 A B C Sandeel 1 2 3 Time (min) C 1 2 3 Time (min) Signal (cps) 6 3 A B Blue whiting C Signal (cps) 2 1 A B Atlantic herring C Signal (cps) 14 7 1 2 3 A B Time (min) Mixed oil 1 2 3 12 1 8 6 y = 16.99x - 781.7 R² =.9939 Time (min) 1 2 3 C Time (min) 4 2 2 4 6 8 1 12 Sele et al., Anal Bioanal Chem, 212
Structural identification by GC-MS/MS Signal (counts) Compound Calculated mass [M+H] + Signal (cps) 4 Found mass [M+H] + 2 m A B C Sandeel Signal (counts) Figure 1. GC-MS1 scan of sandeel oil AsHC-C 15 333.2134 361.2134 ppm 1 2 3 Time (min) Time (min) AsHC-C 17 361.2446 361.2449.83 ppm m/z 316.2 > 15, 16 m/z 344.2 > 15, 16, m/z 388.2 > 15, 16, 159 A AsHC-C 15 AsHC-C 21 45.2134 B 45.2149 3.7 ppm C AsHC-C 17 AsHC-C 21 Time (min) Sele et al., Anal Bioanal Chem, 212
Analysis of arsenolipids in marine oils by reversed-phase HPLC-ICP-MS Fish oil Signal (cps) 3 void AsFAs AsHCs Non-polar As species MeOH 1 Heptane 1% MeOH MeOH 2 Heptane ACN 2 Heptane Red = MeOH1 Black = MeOH2 1 3% MeOH 5 1 15 2 25 3 35 4 45 Time (min) Sele et al., Talanta, 213
Analysis of arsenolipids in marine oils by reversed-phase HPLC-ICP-MS and qtof-ms Signal intensity (cps) 15 1 5 3 AsHC-C 21 9 11 13 15 17 19 21 Time (min) AsHC-C 15 Signal intensity (cps) 2 1 U AsHC-C 17 5 1 15 2 25 3 35 4 45 Time (min) Sele et al., Talanta, 213
Quantitative analysis of arsenolipids in reversedphase HPLC-ICP-MS The signal sensitivity for arsenic increases along an organic gradient. 6 4 3% MeOH Signal (counts) 2 DMA 1% MeOH 1 8 6 4 2 MeOH (%) Ge74 Time-resolved As75 response factor using In115 DMA as calibrant MeOH (%) 1 2 3 Time (min) Ruiz-Chancho et al., 211 Sele et al., Talanta, 213
Arsenolipid profiles in marine oils mg As/kg oil 12. 1. 8. 6. 4. 2.. Photo: NIFES Rest Unknown void sum AsFA AsHC-C17 AsHC-C15 AsHC-C21 Sele et al., Talanta, 213
Parallel coupling of HPLC to both elemental and molecular MS techniques Amayo et al., Anal Chem, 211.
Multi-techniques for identification of novel arsenolipids Amayo et al., 213 OH Trimethyl Arsenic containing fatty alsohols (TMAsFOH)
Toxicity of arsenolipids? Cellular toxicity investigated in cultured human bladder (UROtsa) and liver (HepG2) cells. Cytotoxicity of the arseniccontaining hydrocarbons was comparable to that of arsenite!! => Potential health risk? Foto: NIFES
Arsenic speciation and food safety - a simplified overview Fish Molluscs 1-5 mg/kg 85% 13% 5% 1% 5% 45% 13% 23% 2-1 mg/kg 2% 7% 9% 5% Seaweed 1-1 mg/kg 1% 1% 98% 18% 78% Rice 69% 1% 3% 1% 1% 28%.1-.3 mg/kg 2% toxic (ias) non-toxic (AsB) potentially toxic fat-soluble As arsenosugars other organoarsenicals arsenic fractions potentially toxic J. Feldmann & EM Krupp: Anal Bioanal Chem (211), 399, 1735-1741.
Future food control on As may be more complex than just but maybe also organoarsenic compounds must be taken into account?
Summary ( and perspectives) Arsenic can be found in mg/kg range in marine oil More than 5 different arsenolipids identified Advanced analytical techniques are needed Are the arsenolipids toxic? Food safety concerns? What is the biological role of arsenolipids? - biosynthesis route is still unclear (genetically encoded or just infidelity of biosynthetic pathways) - are arsenolipids restricted to marine organisms? Are arsenolipids only for nerds?