Nonafluoropentanoic acid nfpa

Casein from bovine milk, α-lactose monohydrate, NaOH, sodium-phosphate, and 2-mercaptoethanol were obtained from Sigma-Aldrich (Saint Louis, MO USA). Pancreatin (Pancrex V) was obtained from Paines and Birne (Greenford, UK). Porcine bile extract was obtained from Merck (Darmstadt, Germany). Analytical standards of CML (>99%), CEL (>98%), GH-1 (98.2%) and MG-H1 (>98.2%), as well as the deuterium labelled internal standards CML-D4, CEL-D4, and MG-H1-D3 and G-H1-13 C 2 , were obtained from Iris Biotech (Marktredwitz, Germany). Boric acid (99.5%), chloroform (99.5%), nonafluoropentanoic acid (NFPA; 99%), sodium hydroxide (98%), sodium borohydride (96%), and trifluoroacetic acid (TFA; 99%), were obtained from Sigma (Zwijndrecht, Netherlands). HPLC-grade acetonitrile and methanol were obtained from Actu-all Chemicals (Oss, Netherlands).

Unless otherwise stated, chemicals were purchased from commercial suppliers without further purification: NaCl (≥ 99.5%, Fisher Scientific), NaOH (97%, VWR chemicals), HCl (≥ 37%, VWR chemicals), H 2 SO 4 (> 96%, VWR chemicals), heptafluorobutyric acid (HFBA, 99%, Alfa Aesar), nonafluoropentanoic acid (NFPA, 97%, Sigma Aldrich), pepsin (3839 U/mg protein, Sigma Aldrich), pronase E (4000 PU/mg, Merck), leucine aminopeptidase (18 U/mg protein, Sigma Aldrich), prolidase (553 U/mg protein, Sigma Aldrich), acetonitrile (HPLC-MS/MS grade, VWR), tris-(hydroxymethyl)-aminomethane (TRIS, > 99.9%, Carl Roth), bidistilled water, N-ε-benzoyllysine, CML, [ 2 H 2 ] CML and furosine (PolyPeptide). The following substances were synthesized according to methods stated in the literature: Pyrraline [23, (link)24] (link), MG-H1 [25] (link), CEL [25] (link), maltosine [26] (link), pentosidine [27] (link). The syntheses of [ 13 C 6 , 15 N 2 ] pyrraline, [ 13 C 6 ] MG-H1 and [ 13 C 6 , 15 N 2 ] maltosine were performed in the same manner but using [ 13 C 6 , 15 N 2 ] lysine (pyrraline and maltosine) and [ 13 C 6 ] arginine (MG-H1) instead of the unlabeled amino acids. For the synthesis of [ 13 C 3 ] CEL, [ 13 C 3 ] pyruvic acid was used instead of unlabeled pyruvic acid.

LC-grade H2O (18 MΩ cm) was prepared with a Milli-Q H2O purification system (Millipore, Bedford, MA, USA). Nonafluoropentanoic acid (NFPA), 5-sulfosalicylic acid (SSA), trichloroacetic acid (TCA), anserine, carnosine and LC-MS grade solvents were purchased from Sigma-Aldrich (Milan, Italy).
Tyrosyl-histidine (TH), used as internal standard, and anserine administered to human volunteers were synthesized by Flamma S.p.a. (Chignolo d'Isola, Bergamo, Italy) .