Perchloric acid

Twenty microliter of plasma sample, or 40–50 mg of powdered atrial appendage tissue or 50–70 mg of powdered subcutaneous adipose tissue 120 μl of internal standard (ISTD; 4 μg/ml in H₂O) containing leucine-d3 (CDN Isotopes, D-1973), 40 μl of MilliQ water, 60 μl of 4 M perchloric acid (VWR, CA71007-908) were combined and vortexed. Proteins were precipitated via a 2 min sonication followed by ice bath for 10 min, twice and centrifuged (13,000 RPM, 15 min, 4°C) and the protein pellet was rinsed with 60 μl of 1 M perchloric acid, vortexed and protein precipitated as described above. The new supernatant was combined with the first portion. The sample was split into two 150 μl portions for BCAAs and BCKAs. For BCAAs, 150 μl of extract was neutralized with 60–75 μl of 2 M KOH (VWR, CABH9262-500G) to a pH of 6–10, vortexed and centrifuged (13,000 RPM, 5min, 4°C). The supernatant was transferred to a new tube. For the plasma samples, the precipitate was rinsed with 100 μl of MilliQ water (18Ω), vortexed and centrifuged (13,000 RPM, 5min, 4°C). Supernatants were frozen, freeze dried and reconstituted in 60 μl of 50:50 Methanol (MeOH) water (VWR, CAMX0486-6) to yield 4 μg/ml of internal standard. Samples were derivatized as per the BCAA and BCKA derivatization protocol below as per a prior study (71 (link)–74 (link)).

Potassium tetrachloroplatinate(II) (K₂PtCl₄, ≥99.99%), L-ascorbic acid (≥99%), polyvinylpyrrolidone (Mwt 40000), and commercial Pt/C catalyst E-TEK Pt/C (20% Pt) were purchased from Sigma-Aldrich Chemie GmbH (Munich, Germany). Perchloric acid ((HClO₄), ≥70%), potassium hydroxide (KOH ≥ 85%, pellets), and sodium bicarbonate ((NaHCO₃), MW: 84.01 g/mol) were obtained from (VWR Chemicals BDH).

Standard mix of 22 amino acids, biogenic amines (histamine, putrescine, cadaverine, spermidine, spermine), creatine, creatinine, anserine, hypoxanthine, xanthine and internal standard (IS, 1,7-diaminoheptane) heptafluorobutyric acid (>99%) were obtained from (Sigma-Aldrich, MerckKGaA, Darmstadt, Germany) while acetonitrile and perchloric acid (70–72%) were obtained from VWR (Darmstadt, Germany). Milli-Qsystem (Millipore, Merck KGaA, Darmstadt, Germany) was used for water purification.