Improved conventional and microwave-assisted silylation protocols for simultaneous gas chromatographic determination of tocopherols and sterols: Method development and multi-response optimization

- Comprehensive silylation procedures are illustrated.
- Single protocol was developed for simultaneous analysis of tocopherols and sterols.
- Both conventional and microwave-assisted methods were developed and optimized.
- Temperature and power controlled microwave-assisted derivatizations were compared.
- Various solvents and catalysts enhanced derivatization efficiency.

This paper reports on improved conventional thermal silylation (CTS) and microwave-assisted silylation (MAS) methods for simultaneous determination of tocopherols and sterols by gas chromatography. Reaction parameters in each of the methods developed were systematically optimized using a full factorial design followed by a central composite design. Initially, experimental conditions for CTS were optimized using a block heater. Further, a rapid MAS was developed and optimized. To understand microwave heating mechanisms, MAS was optimized by two distinct modes of microwave heating: temperature-controlled MAS and power-controlled MAS, using dedicated instruments where reaction temperature and microwave power level were controlled and monitored online. Developed methods: were compared with routine overnight derivatization. On a comprehensive level, while both CTS and MAS were found to be efficient derivatization techniques, MAS significantly reduced the reaction time. The optimal derivatization temperature and time for CTS found to be 55 °C and 54 min, while it was 87 °C and 1.2 min for temperature-controlled MAS. Further, a microwave power of 300 W and a derivatization time 0.5 min found to be optimal for power-controlled MAS. The use of an appropriate derivatization solvent, such as pyridine, was found to be critical for the successful determination. Catalysts, like potassium acetate and 4-dimethylaminopyridine, enhanced the efficiency slightly. The developed methods showed excellent analytical performance in terms of linearity, accuracy and precision.