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Protocol for Analyses of Cholesterol and Derivatives in Ocular Tissues Using LC-MS/MS Methods

For the first method, chemical derivatization, the main goal is the detection of protonated cholestadiene peak (m/z 369) by the previous formation of ammonium adducts [M+NH4]+. By this approach it is possible to achieve a simultaneous quantification of FC and CE species using ESI-MS/MS. The acetyl chloride derivatization method has been developed in literature to obtain CE 2:0 without transesterification of the native CE to CE 2:0.

1 Sample pre-processing.

To derivatize:

(a) Prepare a mixture of acetyl chloride/chloroform 1/5 (v/v).

(b) Mix 200 μL of the previous mixture with dried samples (ocular lipid extracts). The lipid extracts will have been previously obtained by processing them by a solid-liquid-liquid extraction method like Bligh and Dyer.

(c) Let the reaction take place for 60 min at room temperature.

(d) After 60 min, remove the reagents by vacuum centrifugation.

Prepare the samples for mass spectrometry by reconstituting the residues in methanol/chloroform (3/1 v/v) 10 mM ammonium acetate and spike the samples with the desired amount of the standard. The samples are now ready for HPLC and mass spectrometry.

2 High-performance liquid chromatography (HPL(c)

(a) Prepare the solvents for HPLC: Solvent A is a mixture of water/methanol (60/40, v/v) containing 10 mM ammonium acetate, and solvent B is a mixture of methanol/ chloroform (3/1, v/v) with 10 mM ammonium acetate.

(b) Mount the column in Accela Autosampler, and equilibrate following the manufacturer's recommendations.

(c) Maintain the column temperature at 45 ℃ and tray temperature at 20 ℃.

(d) Set the flow rate at 260 μL/min and injection volume at 5 μL.

(e) Set the gradient as follows: 35–100% solvent B over 13.0 min, 100% solvent B over 13.0–13.8 min, 100–35% solvent B over 13.8–14.5 min, 35% solvent B over 14.5–18.0 min, and 0% solvent B over 18.0–20.0 min.

3 HESI conditions

(a) Set the spray voltage to 4.4 kV.

(b) Set the capillary at 350 C.

(c) Set the heater at heater at 275 C.

(d) Set S-lens radio frequency (R(f) level to 70.

(e) Set the sheath gas flow rate to 45 units.

(f) Set the auxiliary gas to 15 units.

4 Mass spectrometry

(a) Full scan m/z 150–1500.

(b) Resolution 70,000@ m/z 200.

(c) AGC 1 × 10E6.

(d) Max. ion injection time (IT) 75 ms.

(e) Set MS/MS to data dependent mode, top 10.

(f) Resolution for MS/MS set to 17,500.

(g) AGC for MS/MS set to 2 × 10E5.

(h) Max. IT for MS/MS 75 ms.

(i) Isolation window: m/z 1.3.

(j) Underfill ratio: 0.1%.

(k) Intensity threshold: 1 × 10E3.

(l) Dynamic exclusion time: 3 s.

(m) Normalized Collision Energy (NC(e) 19 ± 5%.

(n) Monitor the SRM transitions in the inclusion list for m/z 446 > 369 for FC and 453 > 378 for D7-FC.

(o) Perform a parent ion scan (PIS) for m/z 369 simultaneously for CE species. This can be achieved by programming a double method.

5 Bioinformatics analysis.

(a) For lipid identification, LipidSearch can be set to look for certain sterol species. Cholesterol ester species are among them. However, peaks are detectable by Xcalibur, via Quan Browser, a Thermo Fisher Scientific software that allows quantification by constructing standard curves using the standards mentioned above. (The Xcalibur quantification method is not detailed her(e). The user can calculate this by other available software in the market.

(b) Lipid identification performed with LipidSearch 4.1 software (Thermo) for cholesterol esters can be achieved using this software. The search criteria were as follows:

  • Mode of searching: Product search.
  • Tolerances:

– Parent m/z tolerance 5 ppm.

– Product m/z tolerance 5 ppm.

– Product ion intensity threshold 1%.

– Filters: top rank, main isomer peak, FA priority.

– Quantification: m/z tolerance 5 ppm, retention time tolerance 0.25 min.

– The following adducts were allowed in positive mode: +H, +NH4, +H–H2O, +H–2H2O, +2H.


  1. Sanjoy K. Bhattacharya. (2019). Metabolomics: Methods and Protocols. Springer-Verlag.
* For Research Use Only. Not for use in diagnostic procedures.
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