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2 protocols using mixed mode solid phase extraction cartridges

1

Aflatoxin B1-Lysine Adduct Quantification

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AFB1-lysine adduct standard was synthesized and purified as previously described by Sabbioni et al. [39 (link)]. Albumin determination reagent (bromocreosol purple), and normal human serum were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). Pronase (25 kU, Nuclease-free) was purchased from Calbiochem (La Jolla, CA). Protein assay dye reagent concentrate and protein standards were purchased from Bio-Rad Laboratories Inc. (Hercules, CA). Boric acid, o-phthaldialdehyde (OPA), 2-mercaptoethanol, FB1 from F. verticilioides (~ 98% purity, TLC), 10× phosphate buffered saline (PBS), ammonium hydroxide, ammonium acetate, sodium chloride, sodium phosphate monobasic, hydrochloric acid, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). OPA reagents were prepared by dissolving 10 mg of OPA and 30 μl of 2-mercaptoethanol in 250 μl of methanol and mixing with 4.75 ml of 3% Boric acid buffer (pH 10.5) and stored at 4 °C avoiding light before use. Mixed mode solid phase extraction (SPE) cartridges, as well as Sep-Pak reversed phase C18 cartridges were purchased from the Waters Corp. (Milford, MA). All other chemicals and solvents were of highest grade and purity available.
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2

Biosynthesis and Purification of AFB1-Lysine Adduct

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AFB1-lysine adduct standard was biosynthesized and purified as described by Sabbioni et al (Sabbioni et al., 1987 (link)). Albumin determination reagent (bromocreosol green) and normal human serum used for normalization were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO), while albumin standard was acquired from Pointe Scientific (Canton, MI, USA). Pronase (25 kU, Nuclease-free) was purchased from Calbiochem (La Jolla, CA). Protein assay dye reagent concentrate was obtained from Bio-Rad Laboratories Inc. (Hercules, CA). Total protein standards, ammonium hydroxide, ammonium phosphate monobasic, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mixed mode solid phase extraction (SPE) cartridges were purchased from the Waters Corp. (Milford, MA). All other chemicals and solvents were of highest grade and purity available.
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