Table 1.
Summary of the methods described in the article.
| Method | Characteristics | Ref. |
|---|---|---|
| Modified Abell-Kendall method | This is a standard reference method for total blood cholesterol measurement. It is a multi-step classical chemical method, which involves saponification of cholesterol esters by hydroxide, extraction with hexane, and color development with acetic anhydride–sulfuric acid. | [28] |
| Fluorometric-enzymatic assay | This assay is based on an enzyme-coupled reaction for determining free cholesterol and cholesterol esters. Esterified cholesterol is converted to cholesterol by cholesterol esterase. The resulting cholesterol is then acted upon by cholesterol oxidase to produce cholest-4-en-3-one and hydrogen peroxide and the corresponding ketone product. Hydrogen peroxide is then detected using sensitive and stable fluorescence probe. | [29] [30] [31] |
| GC/GC-MS: gas chromatography – mass spectrometry | This method is used for routine cholesterol determination. It is used to separate cholesterol from other interfering species. It can be used for determination of free, esterified and total cholesterol levels (based on sample pretreatment). More accurate than classical chemical and enzymatic methods. Extensive sample preparation (including derivatization) prior analysis is required. Labor and resource intensive. | [32] [33] [34] |
| GC-ID-MS | This is a standard reference method for total blood cholesterol measurement. Isotopically labeled cholesterol is added to a sample in an amount approximately equal to the actual amount of analyte present in a sample. Additionally, two calibration mixtures of a known cholesterol and labeled cholesterol concentrations are prepared. Quantification is based on calculating peak area ratio corresponding to unlabeled and labeled cholesterol for unknown sample and comparing it with the ratios calculated for calibration mixtures. | [27] |
| LC/LC-MS: liquid chromatography – mass spectrometry | This method is used for routine cholesterol determination. Similarly to GC, it is used to separate cholesterol from other interfering species. It can be used for determination of free, esterified and total cholesterol level (based on sample pretreatment). More accurate than classical chemical and enzymatic methods. Extensive sample preparation prior analysis is required. Labor and resource intensive. | [35] [36] |
| ESI-MS/MS: electrospray ionization tandem mass spectrometry | This method requires derivatization to increase ionization efficiency. Free cholesterol may be converted to cholesteryl acetate using acetyl chloride. Sample is directly injected to MS. Fragment at m/z 369 is used for quantification (SRM mode and precursor ion scan). | [37] |
| MALDI-MS: matrix-assisted laser desorption ionization-time of flight | This method requires use of matrix. Signal at m/z 369 corresponding to dehydrated molecule [M-H2O+H]+ can be observed. Lipoprotein lipid signals in 369–910 m/z range can be detected in human serum. | [38] |
| MALDI-IM-MS: Matrix-assisted laser desorption/ionization-ion-mobility mass spectrometry | This method takes advantage of ion-mobility to separate chemical noise from the analytes of interest. Coating samples with metal increases the conductivity of insulating substrates, such as tissues or cells. Using sputtered silver nanoparticles instead of matrix provides lower limit of detection based on signals of silver coordinated molecules. Profiling of cholesterol and 7-dehydrocholesterol in SLOS human fibroblast can be done. | [39] |
| DESI-MS: Desorption electrospray ionization mass spectrometry | This method often requires derivatization to increase ionization efficiency. Derivatising reagent can be incorporated into DESI spray. Betaine aldehyde reacts with cholesterol forming charge-labeled cholesterol. | [40] |
| DART: Direct analysis in real time mass spectrometry | This method is suitable for the analysis of non-polar compounds. Signal at m/z 369 corresponding to dehydrated molecule [M-H2O+H]+ can be observed. This method can be used for food and biological samples analysis. Endogenous cholesterol from serum spotted onto the chromatography paper can be desorbed and rapidly ionized. Fast quantitative screening can be achieved. | [26] [41] |
| MALDI-MSI: Matrix-assisted laser desorption/ionization mass spectrometry imaging | This method requires covering tissue section with a thin layer of matrix. It can be used to determine the distribution of endogenous cholesterol sulfate as well as exogenous compounds in human skin tissue sections. Spatial resolution is around 30 μm. | [42] |
| DESI-MSI: Desorption electrospray ionization mass spectrometry imaging | This method allows interrogation of the sample in its native environment. No matrix is required. Reactive DESI (with a betaine aldehyde as a reagent) can be used to image cholesterol in rat brain tissue. Spatial resolution is around 200 μm. DESI can be used to image cholesterol sulfate that is a differentiating compound between healthy and prostate cancer tissue. | [22] [40] |