2.3. Sterol analysis through gas chromatography-mass spectrometry (GC–MS)

Sterol standards used for identification and quantification with GC–MS were cholestanol (5-α-cholestan-3β-ol, ≥95%, Sigma Aldrich), cholesterol (cholet-5-en-3β-ol, ~94%, Sigma Aldrich), stigmasterol (24-ethylcholesta-5,22E-dien-3β-ol, ~95%, Sigma Aldrich), campesterol (24-α-methyl-5-cholesten-3β-ol, ~65%, Sigma Aldrich), sitosterol (24-ethylcholest-5-en-3-β-ol, ≥97%, Sigma Aldrich) and coprostanol (5β-cholestan-3β-ol, ≥98%, Santa Cruz Biotechnology). All standards underwent the BSTFA derivatization at the same time as samples and their TMS-derivatives were used to compare to sample extracts (Table A1). Concentrated samples (run in 30 μL of BSTFA + 10 μL of pyridine) were run with 1 μL injections on an Agilent Technologies 6890N GC with an Agilent DB5-MS capillary column (30 m × 0.32 μm, 0.25 μm film thickness) coupled to a 5973 quadrupole mass spectrometer scanning for masses from 50 to 500 m/z. Operating GC–MS conditions included a temperature program with an initial hold for 1 min at 40 °C ramped to 300 °C at 4 °C/min and held at 300 °C for 24 min (Biache and Philp, 2013). The limit of quantification (0.5 μg/mL) was determined based on linearity of the sterol calibration curves. Final sterol LOQ’s for each particular sample (ng/g of sediment) was dependent on the total mass of sediment weight extracted (Table A2).