Analysis of Underivatized cholesterol

Significance of the topic

Accurate measurement of cholesterol in food and biological samples is critical for nutritional labeling, quality control and research into health impacts. Analysis without derivatization simplifies sample preparation, reduces analysis time and minimizes potential artifacts, making it attractive for routine workflows in food science and clinical laboratories.

Objectives and Study Overview

This study establishes a gas chromatography–flame ionization detection (GC–FID) method for quantifying underivatized cholesterol using 5-α-cholestane as internal standard. The aim is to demonstrate reliable separation, reproducibility and sensitivity under optimized chromatographic conditions.

Methodology and Instrumentation

The analysis was performed on a Shimadzu GC system equipped with an SH-I-5MS capillary column (15 m × 0.25 mm I.D., 0.25 μm film thickness). Key parameters included:

• Oven temperature program A: 200 °C (1 min), ramp 20 °C/min to 330 °C, hold 7.5 min
• Oven temperature program B: isothermal at 300 °C for 10 min
• Injection: 1.0 µL split (1:20) using a single-taper liner with wool, injector at 250 °C, septa purge 5 mL/min
• Carrier gas: helium at constant pressure (67 kPa at 200 °C), linear velocity ≈24 cm/s
• Detector: FID at 340 °C

Key Results and Discussion

The method achieved baseline separation of 5-α-cholestane (internal standard) and cholesterol with sharp, symmetric peaks. Good reproducibility was observed in retention times and peak areas, indicating stable chromatographic conditions. The high detector temperature ensured complete combustion and consistent response, while the use of underivatized cholesterol reduced sample handling steps.

Benefits and Practical Applications

• Simplified sample preparation by eliminating derivatization
• High throughput with short run times and robust separation
• Reliable quantitation using an internal standard to correct for injection variability
• Applicability to food matrices, nutritional studies and routine QC analyses

Future Trends and Applications

Advances may include coupling to mass spectrometry for enhanced specificity and lower detection limits, integration with automated sample prep systems, and miniaturized GC platforms for field testing. Method adaptation for complex matrices and online monitoring could further broaden applications in food safety and clinical diagnostics.

Conclusion

This work presents a robust GC–FID approach for underivatized cholesterol analysis. The straightforward method delivers reliable separation, reproducible quantitation and operational simplicity, supporting its adoption in food science and related fields.

References

Shimadzu Corporation. ERAS-1000-0514, First Edition: Sep. 2023