Robust 9-Minute GC Analysis of Cholesterol
Excellent Sample Throughput with an Rxi-5ms Column
- Highly inert Rxi-5ms column enables analysis of underivatized or derivatized sterol.
- Isothermal approach greatly increases throughput.
- Highly reproducible retention and quantification.
Cholesterol is a vital component in human and animal blood and cell membranes, but cholesterol receives attention primarily because of its association with impaired blood circulation and heart disease. Because of this involvement with cardiovascular disease, many countries require the cholesterol content of food products to be a part of nutritional labels. Much effort is spent on promoting low cholesterol foods and diets and much money is spent on cholesterol-reducing medications [1].
Capillary GC affords qualitative and quantitative analysis of cholesterol and is incorporated into AOAC International methods 970.51E and 976.26.2; According to these methods, cholesterol and other sterols are extracted from the unsaponified fraction of an ether extract of a sample material. The residue is dissolved in chloroform and evaporated (see AOAC methods 933.08 and 970.51A for details [1]). Other methods, including AOAC method 976.26 and AOCS method Ce 3-74 [2,3], describe derivatization of sterols prior to chromatographic analysis.
A highly inert Rxi-5ms capillary column allows qualitative and quantitative analysis of either underivatized or derivatized cholesterol. Figure 1A shows the separation of underivatized cholesterol and internal standard 5-α-cholestane in less than 9 minutes. The temperature program maximizes separation of the analytes and any early-eluting contaminants or extracted matrix interferences encountered when evaluating complex samples. Figure 1B illustrates a rapid, isothermal method that maximizes throughput when interferences are not a concern.
The same analytical conditions can be applied when analyzing derivatized cholesterol, using the standard extraction and derivatization methods referred to above. Figure 2A and Figure 2B show the chromatographic results. In a series of 9 replicates, using the temperature program, the Rxi-5ms column showed both exceptional inertness and excellent reproducibility for either cholesterol or derivatized cholesterol. Table 1 summarizes the results.
If cholesterol analyses are part of your work regimen, we highly recommend using Rxi-5ms columns to ensure reliable results and increase sample throughput.
Table 1 Reproducible results for cholesterol, using an Rxi-5ms column (n=9).
| Analyte | %RSD | |
| Quantitative Data, Area Count | ||
| cholesterol* | 32.5 | 1.78 |
| cholesterol, derivatized** | 17.3 | 1.08 |
| Retention Time | ||
| cholesterol* | 8.030 | 0.008 |
| cholesterol, derivatized** | 8.111 | 0.044 |
*25µg/mL in dimethylformamide.
**50µg/mL in hexane.
Figure 1 Analyze underivatized cholesterol in less than 9 minutes, using an Rxi-5ms column.
| Column | Rxi -5ms, 15 m, 0.25 mm ID, 0.25 µm (cat.# 13420) |
|---|---|
| Standard/Sample | 1,000 μg/mL cholesterol in DMF |
| 1,000 μg/mL 5-α-cholestane in hexane | |
| 25 ng cholesterol, 150 ng 5-α-cholestane on column | |
| Injection | |
| Inj. Vol.: | 1 µL split (split ratio 20:1) |
| Liner: | Splitless taper (4 mm) w/wool |
| Inj. Temp.: | 250 °C |
| Oven | |
| Oven Temp.: | 300 °C (hold 10 min) |
| Carrier Gas | He, constant pressure (9.7 psi, 66.9 kPa) |
| Linear Velocity: | 24 cm/sec @ 200 °C |
| Detector | FID @ 340 °C |
|---|---|
| Notes | Liner cat.# 22405 was used to produce this chromatogram, but has since been discontinued. For assistance choosing a replacement for this application, contact Restek Technical Service or your local Restek representative. |
Figure 2 Results for derivatized cholesterol are equal to those for underivatized cholesterol.
| Column | Rxi-5ms, 15 m, 0.25 mm ID, 0.25 µm (cat.# 13420) |
|---|---|
| Standard/Sample | 1,000 μg/mL cholesterol in hexane |
| 1,000 μg/mL 5-α-cholestane in hexane | |
| 50 ng derivatized cholesterol, 150 ng 5-α-cholestane on column | |
| Injection | |
| Inj. Vol.: | 1 µL split (split ratio 20:1) |
| Liner: | Splitless taper (4 mm) w/wool |
| Inj. Temp.: | 250 °C |
| Oven | |
| Oven Temp.: | 300 °C (hold 10 min) |
| Carrier Gas | He, constant pressure (9.7 psi, 66.9 kPa) |
| Linear Velocity: | 24 cm/sec @ 200 °C |
| Detector | FID @ 340 °C |
|---|---|
| Notes | Liner cat.# 22405 was used to produce this chromatogram, but has since been discontinued. For assistance choosing a replacement for this application, contact Restek Technical Service or your local Restek representative. |
References
[1] www.cholesterol lowdown.org /what_is_cholesterol/index.html
[2] AOAC Official Methods, 15th ed., pp 976-977, 1103-1105 (1990).
[3] AOCS Official Methods and Recommended Practices, 4th ed. (1994).