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Food, Flavors, & Fragrances Article

Robust 9-Minute GC Analysis of Cholesterol

Excellent Sample Throughput with an Rxi™-5ms Column

By Julie Kowalski and Lydia Nolan, Innovations Chemists, and Aaron Muscarella, Innovations Intern
  • 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 details¹). Other methods, including AOAC method 976.256 and AOCS method Ce 3-74²,³, 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.

  1. 5-α-cholestane (IS)
  2. cholesterol

GC_FF00881, GC_FF00882

Column:

Rxi™-5ms, 15m, 0.25mm ID, 0.25µm (cat.# 13420)

Sample:

1000µg/mL cholesterol in DMF, 1000µg/mL 5-α-cholestane in hexane; 25ng cholesterol,
150ng 5-α-cholestane on column

Inj.:

1.0µL, split (20:1), single gooseneck inlet liner w/ wool (cat.# 22405)

Inj. temp.:

250°C

Carrier gas:

helium, constant pressure (9.7psi @ 200°C)

Linear velocity:

24cm/sec.

Oven temp.:

Figure 1A: 200°C (hold 1 min.) to 330°C @ 20°C/min. (hold 7.5 min.) Figure 1B: 300°C (hold 10 min.)

Det.:

FID

Det. temp.:

340°C


Figure 2  Results for derivatized cholesterol are equal to those for underivatized cholesterol.

  1. 5-α-cholestane (IS)
  2. cholesterol

GC_FF00883, GC_FF00884

Column:

Rxi™-5ms, 15m, 0.25mm ID, 0.25µm (cat.# 13420)

Sample:

1000µg/mL cholesterol in hexane, 1000µg/mL 5-α-cholestane in hexane; 50ng derivatized cholesterol,
150ng 5-α-cholestane on column

Inj.:

1.0µL, split (20:1), single gooseneck inlet liner w/ wool (cat.# 22405)

Inj. temp.:

250°C

Carrier gas:

helium, constant pressure (9.7psi @ 200°C)

Linear velocity:

24cm/sec.

Oven temp.:

Figure 2A: 200°C (hold 1 min.) to 330°C @ 20°C/min. (hold 7.5 min.) Figure 2B: 300°C (hold 10 min.)

Det.:

FID

Det. temp.:

340°C

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).