Novel Charge-Switch Derivatization Method Using 3‑(Chlorosulfonyl)benzoic Acid for Sensitive RP-UHPLC/MS/MS Analysis of Acylglycerols, Sterols, and Prenols

Chemical derivatization involves the reaction of an analyte
with
a derivatization agent to modify its structure, improving the peak
shape, chromatographic performance, structural analysis, ionization
efficiency, and sensitivity. A novel derivatization method using 3-(chlorosulfonyl)benzoic
acid is developed for the determination of monoacylglycerols, diacylglycerols,
free sterols, and tocopherols using the reversed-phase ultra-high-performance
liquid chromatography–tandem mass spectrometry (RP-UHPLC/MS/MS)
method in the negative ion mode. The chromatographic and mass spectrometric
properties of derivatized lipids are investigated by using 29 lipid
standards spanning four lipid classes. The derivatization process
is optimized using pooled plasma spiked by 9 internal standards, achieving
an optimal yield with a reaction time of 40 min at 60 °C. The
stability of the derivatives is confirmed, with short-term stability
maintained for 10 h at 4 °C and long-term stability preserved
for 5 days at −80 °C. The repeatability and reproducibility
are verified by one/two operator(s), which underscores the simplicity
and robustness of the method, and calibration curves with high linear
regression coefficients illustrate the accuracy of the method. The
derivatization approach, which combines RP-UHPLC/MS/MS and the use
of specific fragmentation patterns, significantly reduces limits of
detection, reaching 15–25 pmol/mL for free sterols in plasma.
The optimized method is applied to the analysis of human plasma, leading
to the identification of 92 lipid species in the targeted lipid classes.
This represents a substantial improvement in sensitivity and detection
capabilities compared to those of previously reported methods.