O phthaldialdehyde opa

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.

GTP used for the study was obtained from the US-China joint venture Shili Natural Product Company, Inc. (Guilin, Guangxi, China) and the purity of GTP is higher than 98.5% according to the analysis by Guangxi Standard Bureau. Boric acid, D-erythro-sphingosine, D-erythro-sphinganine, FB₁ from F. verticilioides (approximately 98% by HPLC), formic acid, hydrochloric acid, 2-mercaptoethanol, O-phthaldialdehyde (OPA), 10 x phosphate-buffered saline (PBS), and sodium phosphate monobasic were purchased from Sigma-Aldrich (St. Louis, MO, USA). D-erythro-C₂₀-sphingosine was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Triethylammonium formate HPLC buffer (pH 3.0) was purchased from Regis Technologies, Inc. (Morton Grove, IL, USA). Other HPLC-grade solvents including acetonitrile, ethyl acetate, methanol, 2-propanol, and water were from Honeywell Burdick & Jackson (Muskegon, MI, 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).

Fresh samples of stone fish (A. lecanora) were locally supplied from Kedah and Langkawi breeding centers in Malaysia. The internal organs were immediately removed, and samples were washed and transported in iced bags to the laboratory. The samples were rewashed, packed in plastic bags and stored at −80 °C until use. Prior to analysis, the samples were freeze dried and grounded with a Waring^® blender (Stamford, CT, USA), sieved through 600 µm wire gauze and kept at −40 °C until analysis. Bromelain from pineapple stem tissue, 100 gr (2.4 to 3 U/mg International Pharmaceutical Federation (FIP)) was obtained from Acros Organics (Geel, Belgium). Angiotensin converting enzyme (≥2.0 units/mg protein) and N-Hippuryl-His-Leu hydrate-powder, (≥98%) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dithiothreitol and O-phthaldialdehyde (OPA), 97% were obtained from Sigma–Aldrich (Buchs, Switzerland). All other chemicals used for this study were analytical grade and obtained from Acros organics (Geel, Belgium), Fisher Scientific (Loughborough, Leics, UK), J.T Baker (Bangkok, Thailand) and Merck KGaA (Darmstadt, Germany).