Esi probe

Pigments were extracted from fresh algal cells with methanol. The identification and quantification of carotenoids were performed using an ACQUITY Ultra Performance Liquid Chromatography (UPLC) H-Class coupled in line to a Xevo TQ-XS triple quadrupole mass spectrometer equipped with an ESI probe (Waters, Milford, MA, USA). Samples were injected into an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters), and the eluents were acetonitrile (A), methanol (B), and demineralized water containing 0.1% (v/v) formic acid (C). The gradient was initiated from 58% A, 27% B, and 15% C, then transiently to 92% A and 8% C at 4 min, and finally reverted to its initial composition at 8 min, followed by an equilibration phase of 2 min. The flow rate was at 350 μL min⁻¹. Nitrogen was used as desolvation gas (800 L/h at 400 °C). The spray capillary voltage was 3000 V for the positive ion mode. Multiple reaction monitoring (MRM) scanning mode was used for mass spectrometry detection. Ions used for the quantification of fucoxanthin, diadinoxanthin, and diatoxanthin were [M + H]⁺ -> 109.1, [M + H]⁺ -> 91.0, and [M + H]⁺ -> 105.1, respectively. The standards employed for the identification and quantification of carotenoids were purchased from Sigma (St. Louis, MO, USA).

Merck silica gel (100–200 mesh) column chromatography Sigma Aldrich, St. Louis, MO, USA and preparative thin-layer chromatography Sigma Aldrich, St. Louis, MO, USA were used to purify synthesized compounds. Compounds were identified and characterized using ¹H NMR and ¹³C NMR (Bruker DPX-300, Bruker, Manning Park Billerica, MA, USA), IR (Perkin-Elmer 1640 FT-IR, PerkinElmer, Waltham, MA, USA) and Mass (WATERS Micro-mass ZQ 4000; ESI Probe, Waters, Milford, MA, USA) spectrometers. A Perkin-Elmer 343 Polari meter (PerkinElmer, Waltham, MA, USA) was used to measure optical rotations. A Buchi B-540 melting point apparatus (Sigma Aldrich, St. Louis, MO, USA) was used to record melting points. Reactions were conducted under dry nitrogen atmospheric conditions. Solvents were distilled prior to their use.

Pigments were extracted from fresh algal cells using methanol. By measuring the absorbance, respectively, at 664 nm and 630 nm with a spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA), chlorophyll concentration was calculated with the following equation [47 (link)]:

Identification and quantification of fucoxanthin were performed on ACQUITY Ultra Performance LC H-Class coupled in-line to a Xevo TQ-XS triple quadrupole mass spectrometer with a ESI probe (Waters, Milford, Massachusetts, USA). Samples were injected on a ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters), and the eluents were acetonitrile (A), methanol (B) and demineralized water contained 0.1% (v/v) formic acid (C). The gradient was initiated from 58% A, 27% B, and 15% C, then transiently to 92%A and 8% C at 4 min, and finally reverted to its initial composition at 8 min followed by an equilibration phase of 2 min. The flow rate was at 350 μL/min. Nitrogen was used as desolvation gas (800 L/h at 400 °C). The spray capillary voltage was 3 kV for the positive ion mode. Multiple reaction monitoring (MRM) scanning mode was used for mass spectrometry detection. The standards of fucoxanthin were purchased from Sigma (St. Louis, MO, USA).