Ammonium hydroxide

The Chinese dry-cured hams were prepared and sampled in Jinnian Ham Co., Ltd. (Jinhua, China). After slaughtered and stored at 4 °C for 48 h, six trimmed pig hind legs with an average weight of 14.5 ± 0.5 kg (pH= 5.9 ± 0.2) were used for the preparation of Chinese dry-cured fatty hams (FH) and lean hams (LH). The ammonium acetate, ammonium hydroxide, methanol, and acetonitrile were bought from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA) and China National Medicines Co., Ltd. (Beijing, China).

Cellulose acetate (CA) powder (average Mn~30,000 by GPC) was supplied by Sigma Aldrich (Burlington, MA, USA). DMF (99%, Honeywell, Charlotte, NC, USA) was used for polymer dissolution. Ferric chloride hexahydrate (FeCl₃ * 6H₂0), iron sulfate (Fe(SO₄)₂ * 6H₂O) (Fluka, Buchs, Switzerland), sodium hydroxide and ammonium hydroxide (Merck, Rahway, NJ, USA) were used for the iron nanoparticle synthesis. Calcium chloride (94%, Roth, Karlsruhe, Germany), hydrochloric acid (37%, Sigma Aldrich, Burlington, MA, USA), tris(hydroxymethyl)aminomethane (99.8%, Sigma Aldrich, Burlington, MA, USA) and anhydrous disodium phosphate (99%, Sigma Aldrich, Burlington, MA, USA) were employed for the mineralization studies. All reagents were analytical grade and were used without further purification. The water utilized in all experiments was distilled water.

Acetic acid (CAS No. 64-19-7), ammonium hydroxide (CAS 1336-21-6), benzyl isocyanate (CAS 3173-56-6) or trimellitic anhydride (TMA, CAS 552-30-7), capsaicin (CAS 404-86-4), formaldehyde (CAS 50-00-0), toluene (CAS 108-88-3), lactose (CAS 63-42-3), and salicylic acid (CAS 69-72-7) were either purchased from Millipore Sigma (St. Louis, MO, USA) or Merck KGaA (Darmstadt, Germany). Triton™ X-100 (CAS 9002-93-1) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sodium dodecyl sulfate (SDS, CAS 151-21-3) was purchased from Carl Roth (Karlsruhe, Germany). Due to issues with chemical stability and compatibility with tissue culture materials, benzyl isocyanate was replaced with trimellitic anhydride for the A549 cell model. The test chemicals were supplied with appropriate certificates of analysis indicating their purity. The identities of the test chemicals were masked during study execution, data collection, and analysis and were unmasked for finalizing the study report.

Polar metabolites were separated on a Sequant ZIC-pHILIC HPLC column (100 mm × 2.1 mm × 5 µm particle size) at 50 °C using the following gradient: 95% mobile phase B from 0–2 min, decreased to 30% B over the next 16 min, held at 30% B for 8 min, then increased to 95% B over the next 1 min, then held at 95% B for the next 8 min. The flow rate was 130 µl per min. For each analysis, 2 µl per sample was loaded onto the column. Mobile phase A consisted of 10 mM ammonium acetate in 10% (v/v) ACN and 90% (v/v) water with 0.1% (v/v) ammonium hydroxide (Sigma-Aldrich). Mobile phase B consisted of 10 mM ammonium acetate in 95 (v/v) ACN and 5% (v/v) water with 0.1% ammonium hydroxide.
The LC system (Vanquish Binary Pump, Thermo Scientific) was coupled to a Q-Exactive HF Orbitrap mass spectrometer through a HESI II source (Thermo Scientific). Source and capillary temperatures were 350 °C, the sheath gas flow rate was 45 units, the aux gas flow rate was 15 units, sweep gas flow rate was 1 units, spray voltage was 3.0 kV for both positive and negative modes and the S-lens RF was 50.0 units. The MS was operated in a polarity switching mode, with alternating positive and negative full-scan MS and MS2 (top 10). Full-scan MS were acquired at 60,000 resolution (at 200 m/z) with a 1 × 10⁶ AGC target, max ion accumulation time of 100 ms and a scan range of 70–900 m/z. MS2 scans were acquired at 45,000 resolution (at 200 m/z) with 1 × 10⁵ AGC target, max ion accumulation time of 100 ms, 1.0 m/z isolation window, stepped NCE at 20, 30 and 40, and a 30.0 s dynamic exclusion.
LC–MS files for metabolomics were processed using Compound Discoverer 3.3 (Thermo Scientific) in a discovery mode workflow. All peaks between 0 and 22 min retention time and 0–5,000 Da MS1 precursor mass were grouped into distinct chromatographic profiles (that is, compound groups) and aligned against the reference file (the QC file running in the middle of all the files). Profiles not reaching a minimum peak intensity of 5 × 10⁴, a maximum peak width of 3 min, a signal-to-noise ratio of 1.5 and a fivefold intensity increase over blanks were excluded from further processing. Profiles having fewer than five points across the peak were also excluded. Element compositions were predicted with 5 ppm mass tolerance based on MS1 precursor mass. Precursors were matched to compounds by searching against databases including Biocyc, Human metabolome database and Kyoto Encyclopedia of Genes and Genomes. MS/MS spectra were searched against mzCloud (Thermo Scientific) containing 19,503 unique molecular compositions, mzVault libraries including in-house curated MS2 spectra of 151 standards, 598 polar compounds from Bamba lab, the Fiehn lab HILIC library of 3,061 entries, the KI-GIAR zic HILIC library of 814 entries and six other libraries from MassBank of North America. The resulting features were filtered based on the peak quality rating and only the features that had a peak rating greater than 4.0 (on a scale of 0–10) in at least 20 samples were kept for further analysis. Compound annotation was done manually by examining the formula composition, MS2 spectrum and retention time similarities to library entries. Only features with instrument QC coefficient variance below 30% were kept for further analysis.