Esquire hct ultra

LC-MS/MS was conducted using an Ultimate 3000 nano-LC system connected to either an LTQ Orbitrap Elite (Thermo) or Esquire HCT Ultra ion trap (Bruker), both as previously described (36 (link)).

The cPE extract was first analyzed by HPLC/MS and the major compounds present in this fraction isolated by semi preparative HPLC to yield compounds (1) to (7). Dried extracts were solubilized in methanol at 10 mg/ml for analytical and semi preparative experiments, the injected volume being different, and filtered on a 0.45 μM PTFE filter before HPLC analysis. The cPE samples were analyzed by HPLC-UV-DAD (Waters 2695) at different wavelengths, using a RP Nucleodur C18ec column (250 by 4.6 mm, 5 μm particle size, Macherey Nagel) column and using water plus formic acid 0.05% as solvent A, and acetonitrile as solvent B plus formic acid 0.05%, and coupled with mass spectrometry (MS) using an ion trap Bruker Esquire HCT Ultra mass spectrometry instrument equipped with an electrospray ion source in positive and negative mode (data were viewed by using Hystar Bruker software). The analytical conditions were optimized to enhance the separation of the compounds present in this fraction. An isocratic method (solvent A: 20%, solvent 80B: %, flow rate: 1.5 ml min⁻¹) was chosen. HPLC quality solvents were purchased from Fischer Chemicals (Leicestershire, United Kingdom).

One unidentified pigment in Nephroselmis sp. was also analyzed by mass spectrometry (MS) analysis using an ion trap Bruker Esquire HCT Ultra MS instrument equipped with an electrospray ion source in positive mode (data were viewed by using Hystar Bruker software). HPLC quality solvents were purchased from Fischer Chemicals (Leicestershire, UK). Dried extract of Nephroselmis sp. HL was dissolved in methanol/acetone 50:50 (v/v) at 5 mg·mL⁻¹. Optimized pseudo isocratic elution was applied on a RP C18ec Macherey Nagel Nucleodur C18ec (4.6 by 250 mm) column and using as solvent A, water plus formic acid 0.05%, and as solvent B (methanol plus formic acid 0.05%). The analytical conditions were as follows: Flow rate one mLPmin⁻¹, injection volume of 50 µL, 10 min 5% of A to 0% of A, then 35 min 100% of B.
Siphonaxanthin characterization (Figure 1): UV/VIS (ethanol) λmax (retention time): 267, 454 nm (6.7 min), MS-ESI + m/z: 623.4 [M + Na]⁺; 601.4 [M + H]⁺; and 583.4 [M + H-H₂O]⁺.