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Cholesterol UNIT D1.

This unit describes the methods for quantitative determination of cholesterol in food and
foodstuffs by chromatography and by an enzymatic determination using spectro-
photometry. Two types of lipid classes including free cholesterol and cholesterol ester
exist in food. Total cholesterol is measured after chemical hydrolysis of cholesterol esters
to free cholesterol. In a similar way, fatty acids can be analyzed by gas chromatography
(GC). Although successful results can be obtained by high-performance liquid chroma-
tography (HPLC) without any derivatization of cholesterol, more sensitive and specific
determination can be achieved by chemical conversion of cholesterol to the corresponding
derivatized form. Since food and foodstuffs contain cholesterol and many sterol analogs,
including phytosterols and oxidized cholesterol, appropriate chromatographic separation
of cholesterol itself should be required for accurate quantification. The HPLC system
required for cholesterol quantification is widely available in laboratories; however, it may
be difficult to obtain a complete isolation of cholesterol by HPLC. GC is the most adequate
method for this purpose and has been generally applied for quantitative measurement of
cholesterol. Trimethylsilyl (TMS) ether derivatization is necessary before GC analysis.
Colorimetric determination of cholesterol by chemical coloration of anisidine has been
widely used and is one of the traditional methods. However, this measurement is not
specific to cholesterol but also detects other corresponding analogs, and is therefore not
covered in this unit. Enzymatic measurement is more specific to cholesterol compared to
the colorimetric method, and expensive analytical instruments are not required. Lipid
extraction prior to determination is not required in the enzymatic analysis. However, the
results obtained by enzymatic measurement for certain foodstuffs are usually overesti-
mated compared to those obtained by other chromatographic methods.
In the Basic Protocol, gas-liquid chromatography (GLC) using an open tubular wall,
coated, fused silica column with nonpolar liquid phase is described. In Alternate Protocol
1, reversed-phase HPLC (RP-HPLC) is applied for separation and quantification of
cholesterol. In Alternate Protocol 2, enzymatic measurement is applied for determination
of cholesterol.

MEASUREMENT OF CHOLESTEROL BY GAS CHROMATOGRAPHY BASIC


PROTOCOL
Prior to instrumental analysis, cholesterol is extracted from the food sample (UNIT D1.1)
and saponified. The amount of the food sample depends on the cholesterol content; a
sample containing ∼1 mg of cholesterol is required for lipid extraction. Saponification
hydrolyzes the carboxyl group attached to carbon in cholesterol and yields only free
cholesterol in unsaponifiable matter. The unsaponifiable matter can be extracted by
n-hexane, diethyl ether, or petroleum ether. Determination of cholesterol by GLC requires
derivatization of the polar hydroxyl group of cholesterol to a nonpolar group such as
trifluoroacetyl, acetyl, or TMS ether. This conversion of cholesterol allows high resolution
of cholesterol from other sterol analogs by GLC. In the present protocol, unsaponifiable
matter is extracted using n-hexane, and TMS ether derivatization of cholesterol is
performed. This protocol requires prior knowledge of GLC methodologies and adherence
to instructions from the instrument’s manufacturer.
Materials
Food sample
5α-cholestane solution (internal standard; see recipe)
Methanol
Chloroform
Lipid Composition
Contributed by Toshiaki Ohshima D1.3.1
Current Protocols in Food Analytical Chemistry (2001) D1.3.1-D1.3.14
Copyright © 2001 by John Wiley & Sons, Inc.
0.88% (w/v) KCl solution
Anhydrous sodium sulfate
0.5 M ethanolic potassium hydroxide solution (see recipe)
n-Hexane
Nitrogen gas
Pyridine
Hexamethyldisilazane (HMDS)
Trimethylchlorosilane imidazole (TMCS)
Cholesterol solutions (standard; see recipe)
Homogenizer with high-speed rotary blades
Vacuum filtration device
Whatman no. 1 filter paper
Separatory funnel
50-ml glass, tapered, round-bottom flask
Rotary evaporator
Condenser
60°C water bath
5-ml screw-cap glass vials
Gas chromatograph (GC) with:
Flame ionization detector
Open tubular-wall column (0.25-mm i.d. × 30-m) coated with OV-1 equivalent
liquid phase (0.25-µm film thickness)
Helium as a carrier gas
Digital integrator
NOTE: All organic solvents should be reagent grade. HMDS and TMCS should be kept
in a desiccator.

Extract total lipids


1. To an aliquot of food sample containing ∼1 mg cholesterol, add 1 ml working
5α-cholestane solution (internal standard) followed by 10 vol methanol and homoge-
nize for 5 min.
Semi-solid and pasty samples must be sufficiently homogenized. Solid samples should be
crushed and quantitatively passed through a chemical sieve with a <0.2-mm mesh.
2. Add 20 vol chloroform and homogenize again for 3 min.
3. Vacuum filter the homogenate through a Whatman no.1 filter paper and collect the
filtrate fraction.
4. Add 10 vol methanol and 20 vol chloroform to the residue cake and homogenize 3
min.
5. Filter the homogenate as in step 3 and pool the second filtrate with the first.
6. Add 1⁄4 vol 0.88% KCl solution and allow to separate into two layers in a separatory
funnel.
7. Collect the lower (chloroform) layer containing total lipids and dry over anhydrous
sodium sulfate.

Saponify total lipids


8. Transfer the chloroform solution of total lipids to a 50-ml glass, tapered, round-bot-
tom flask. Remove chloroform under vacuum using a rotary evaporator at 30°C.
Cholesterol

D1.3.2
Current Protocols in Food Analytical Chemistry
9. Add 20 ml of 0.5 M ethanolic potassium hydroxide solution.
10. Connect the flask to a condenser and reflux 30 min in a 60°C water bath to saponify
the total lipids.

Extract unsaponifiable matter


11. Cool the refluxed solution to room temperature, remove the condenser, and transfer
the saponified solution to a separatory funnel.
12. Add 60 ml water to dilute the alcohol, and subsequently extract the unsaponifiable
matter three times with 20 ml n-hexane.
When the amount of water added is small and the alcohol concentration in the saponified
solution is high, fatty acid potassium salts may transfer into the ether layer along with
unsaponifiable matter, including cholesterol. For quantitative recovery of cholesterol, as
well as the complete separation of the fatty acid potassium salts from the cholesterol
fraction, lowering the alcohol concentration by adding excess water prior to ether
extraction is recommended. When the alcohol concentration is sufficiently low, the saponi-
fied solution forms easily.
13. Pool the n-hexane extracts and wash three times with 1 vol water. Dry over anhydrous
sodium sulfate.

Prepare TMS ether derivative


14. Reduce the volume of n-hexane in vacuo with a rotary evaporator at 30°C and transfer
the solution to a 5-ml screw-cap glass vial.
15. Dry the unsaponifiable matter under a nitrogen stream and add the following:
0.5 ml pyridine
0.2 ml HMDS
0.1 ml TMCS.
16. Purge the head space using the nitrogen stream and screw the cap on tightly.
17. Incubate 30 min at ambient temperature to complete silylation.

Perform GLC
18. Remove excess pyridine under nitrogen at room temperature and add 1 ml n-hexane
(final ∼1 mg/ml cholesterol).
19. Inject in duplicate up to 2 µl n-hexane solution into the GC. Perform GLC using the
following analytical conditions:
Injection port and detector block: 330°C
Column oven temperature program: 250° to 320°C at 4°C/min
Column inlet helium pressure: 1.75 kg/cm2 at 250°C
Split ratio: 60:1.
Under these analytical conditions, 5α-cholestane and the TMS ether derivative of choles-
terol will be eluted at ∼16 and 20 min, respectively.
20. Determine the peak areas of cholesterol and the internal standard with a digital
integrator.

Run cholesterol standards


21. For each cholesterol standard concentration, prepare a mixture of 1 ml working
5α-cholestane solution (internal standard) and 1 ml working cholesterol solution
(standard) in a 5-ml screw-cap glass vial.
Lipid Composition

D1.3.3
Current Protocols in Food Analytical Chemistry
22. Subject to silylation (steps 15 to 17) and analyze (steps 18 to 20).

Analyze data
23. Divide the area ratio of cholesterol in the samples by that of the internal standard to
obtain a standard response ratio.
24. Plot the average response ratio against the ratio of cholesterol to internal standard in
the standards.

ALTERNATE HPLC MEASUREMENT OF CHOLESTEROL


PROTOCOL 1
Prior to HPLC determination, total lipids, including cholesterol, are extracted from food
and foodstuffs. As in the Basic Protocol, the amount of the food sample depends on the
cholesterol content; a sample containing ∼1 mg of cholesterol is required. Free fatty acids
are completely removed from the cholesterol extract, and a benzoate ester derivative of
cholesterol that has a specific UV absorbance at 230 nm is separated by RP-HPLC. This
protocol allows accurate determination of cholesterol in food at levels as low as 10 ng
benzoate derivative. This protocol requires prior knowledge of HPLC methodologies and
adherence to instructions from the instrument’s manufacturer. A typical result is shown
in Figure D1.3.1.

Additional Materials (also see Basic Protocol)


HPLC-grade methanol
Saturated NaCl solution
Petroleum ether
Dry pyridine
Benzoyl chloride
0.1 M HCl
Diethyl ether
0.1 M Na2CO3
Cholesterol (>99% purity)
80°C water bath
60°C vacuum oven
High-performance liquid chromatograph (HPLC) with:
Pump
Sample injector
Reversed-phase HPLC column: ODS column, 3.9-mm i.d. × 30-cm length,
10-µm particle size
UV detector (230 nm)
Digital integrator
NOTE: All organic solvents should be reagent grade. Diethyl ether should be kept at 4°C.

Extract and saponify lipids


1. Extract total lipids as described (see Basic Protocol, steps 1 to 7), but use HPLC-grade
methanol and do not include 5α-cholestane as an internal standard.
2. Saponify lipids as described (see Basic Protocol, steps 8 to 10).

Remove free fatty acids


3. Add 10 ml n-heptane, reflux for another 2 to 3 min, and cool to room temperature.
4. Quantitatively transfer the refluxed solution to a separatory funnel and add 5 ml
saturated NaCl solution.
Cholesterol

D1.3.4
Current Protocols in Food Analytical Chemistry
5. Extract the aqueous phase twice with 10 ml petroleum ether.
6. Combine petroleum ether extracts and wash with 5 vol water. Dry over anhydrous
sodium sulfate.
When the amount of water added is small and the alcohol concentration in the saponified
solution is high, fatty acid potassium salts may transfer into the ether layer along with
unsaponifiable matter, including cholesterol. For quantitative recovery of cholesterol, as
well as the complete separation of the fatty acid potassium salts from the cholesterol
fraction, lowering the alcohol concentration by adding excess water prior to ether
extraction is recommended. When the alcohol concentration is sufficiently low, the saponi-
fied solution forms easily.
7. Transfer the petroleum ether solution to a 50-ml glass, tapered, round-bottom flask
and evaporate in vacuo using a rotary evaporator at 30°C.

100%
Response

cholesterol

2 4 6 8 10 12
Time (min)

Lipid Composition
Figure D1.3.1 A typical HPLC chromatogram of cholesterol benzoate derived from dried egg yolk.
D1.3.5
Current Protocols in Food Analytical Chemistry
Prepare benzoate ester derivative
8. Transfer the solution to a 5-ml screw-cap glass vial.
9. Add 4 ml dry pyridine and 0.2 ml benzoyl chloride and stir for 5 min at room
temperature.
10. Incubate 20 min in an 80°C water bath.
11. Pour the reaction mixture into a separatory funnnel, and add 50 ml of 0.1 M HCl and
50 ml diethyl ether to extract cholesterol benzoate.
12. Wash diethyl ether extract with 50 ml of 0.1 M HCl to remove excess benzoyl
chloride.
13. Wash with 50 ml water.
14. Wash with 50 ml of 0.1 M Na2CO3 to neutralize the extract.
15. Wash again with water to remove excess carbonate.
16. Transfer to a beaker and evaporate to dryness under a nitrogen stream. Place the
beaker in a vacuum oven at 60°C and dissolve the dried extract in 1 ml chloroform.

Prepare standards
17. Convert 100 mg cholesterol to benzoate derivative as above (steps 8 to 16).
Commercially available authentic cholesterol usually absorbs moisture and exists as a
monohydrate. Drying the authentic cholesterol at 80°C for 2 hr prior to use is recom-
mended.
18. Prepare at least three different concentrations (∼1 mg/ml) in duplicate.

Perform RP-HPLC
19. Inject 20 ìl sample and standards into the HPLC and analyze using the following
analytical conditions:
HPLC column: ODS, 3.9-mm i.d. × 30-cm length, 10-µm particle size
Mobile phase: methanol at 2 ml/min
Detector: UV at 230 nm.
20. Prepare a calibration curve by plotting average detector response (peak area) of the
standards versus cholesterol concentration.
21. Use the peak area of the sample and the calibration curve to determine the amount
of cholesterol in the sample.

ALTERNATE ENZYMATIC MEASUREMENT OF CHOLESTEROL


PROTOCOL 2
Test combination kits for enzymatic determination of cholesterol in food are now
commercially available. For the determination of total cholesterol, esterified cholesterol
is hydrolyzed to free cholesterol and fatty acid under mild alkaline conditions. Cholesterol
oxidase oxidizes free cholesterol to cholest-4-en-3-one to generate hydrogen peroxide,
which further oxidizes methanol to formaldehyde. Formaldehyde then reacts with acetyl
acetone in the presence of NH4+ ions to form yellow lutidine dye, which is subsequently
determined spectrophotometrically.

Cholesterol

D1.3.6
Current Protocols in Food Analytical Chemistry
Materials
Food sample
Sea sand
1.0 M methanolic potassium hydroxide solution (see recipe)
Isopropanol
Test combination F-kit for cholesterol determination (R-Biopharm), containing:
Bottle 1:220,000 U catalase in 95 ml ammonium phosphate buffer, pH 7.0, and
2.6 M methanol (store up to 3 months at 4°C)
Bottle 2: 0.05 M acetylacetone/0.3 M methanol (store up to 3 months at 4°C)
Bottle 3: 12 U cholesterol oxidase suspension in a 0.8-ml volume (store up to
1 yr at 4°C)
Bottle 4: 1.00 mg/ml cholesterol standard in isopropanol (store up to 3 months
at 4°C)
50-ml volumetric flask
60°C and 37° to 40°C water baths
Reflux condenser
Whatman no. 5A filter paper
Brown glass bottle
10-ml glass test tubes
Glass cuvette with 1-cm light path
Spectrophotometer
NOTE: All organic solvents should be reagent grade.

Prepare sample
1. Accurately weigh ∼1 g food sample into a 50-ml volumetric flask and add 1 g sea
sand to complete saponification (step 3).
Semi-solid and pasty samples must be sufficiently homogenized. Solid samples should be
crushed and quantitatively passed through a chemical sieve with a <0.2-mm mesh. If the
sample solution includes colorants, it should be treated with activated charcoal (e.g., with
Clarocarbon F from Merck) at 5% of the sample weight.
2. Add 20 ml freshly prepared 1.0 M methanolic potassium hydroxide solution and 10
ml isopropanol.
3. Heat in a water bath at 60°C under a reflux condenser for 30 min while stirring with
a magnetic stirrer.
4. Allow the turbid solution to cool. Remove the magnetic stir bar and bring to 50 ml
with isopropanol at room temperature.
5. Mix the fluid and filter through Whatman no. 5A filter paper. Retain the clear sample
solution for the assay.

Perform assay
6. Mix three parts catalase (bottle 1) with two parts acetylacetone/methanol (bottle 2)
in a brown glass bottle. Allow to stand at room temperature 1 hr before use.
This solution can be prepared in advance and stored up to 3 months at 4°C. Again, it should
be allowed to stand at room temperature for 1 hr before use.
7. Prepare a sample blank (without cholesterol oxidase) by pipetting 5 ml catalase/ace-
tylacetone/methanol solution and 0.4 ml sample solution into a 10-ml glass test tube.
8. Prepare the test sample by transferring 2.5 ml from the sample blank to a new tube
and adding 0.02 ml cholesterol oxidase from bottle 3. Lipid Composition

D1.3.7
Current Protocols in Food Analytical Chemistry
9. Mix thoroughly, cover the tubes, and incubate 60 min in a 37° to 40°C water bath.
10. Allow to cool to room temperature. Read absorbance of the sample blank and then
the test sample in the same 1-cm glass cuvette against air at 405 nm.
11. Subtract absorbance of the blank from the absorbance of the sample. Determine
concentration using the simplified equation c (g/liter) = 0.711 × ∆A405.
This equation is derived from the equation:

 V × mol. wt. × DF 
c=  × ∆A405
 ε × d × v × 100 mmol/mol 
where V is final volume (5.4 ml), v is sample volume (0.4 ml), mol. wt. is the molecular
weight of the substance to be assayed (386.64 g/mol), DF is the dilution factor (2.52/2.5
= 1.008), d is the light path (1 cm), and ε is the extinction coefficient of lutidine dye at 405
nm (7.4 liter/mmol/cm).

REAGENTS AND SOLUTIONS


Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

5α-Cholestane solution
Prepare a 1.0 mg/ml 5α-cholestane (>99% pure) stock standard solution in n-hex-
ane. Store up to 3 months at 4°C. Prepare a working solution by diluting the stock
solution to 0.2 mg/ml in dimethylformamide (DMF). Prepare fresh.
Cholesterol solutions
Prepare a 1.0 mg/ml cholesterol (>99% pure) stock standard solution in dimethyl-
formamide (DMF). Store up to 1 week at 4°C. Prepare working solutions by diluting
the stock solution with DMF to obtain concentrations ranging between 0.05 and 0.5
mg/ml. Prepare fresh.
Commercially available authentic cholesterol usually absorbs moisture and exists as mono-
hydrate. Drying the authentic cholesterol at 80°C for 2 hr prior to use is recommended.
Ethanolic potassium hydroxide solution, 0.5 M
Dissolve 5.6 g KOH in 2 ml water
Add 100 ml ethanol
Store up to 1 week at 4°C
Methanolic potassium hydroxide solution, 1.0 M
Dissolve 11.2 g KOH in 2 ml water
Add 100 ml methanol
Prepare fresh

COMMENTARY
Background Information of short-wavelength UV light (200 to 210 nm;
The TMS ether derivatives of sterols (in- Duncan et al., 1979; Carrol and Rudel, 1981).
cluding cholesterol) and their oxidation prod- Most organic compounds absorb UV light in
ucts in foods are well resolved by GLC. The this area of the spectrum, and therefore detec-
TMS ether derivatives are eluted in the follow- tion by UV light at ∼205 nm is not specific to
ing order: cholesterol, campesterol, stigma- cholesterol itself. Thus, the Basic Protocol pro-
sterol, and β-sitosterol. vides a more accurate determination of choles-
Detection of cholesterol in extracted total terol in food matrix. Triglycerides consisting of
lipids by HPLC is usually based on absorbance three molecules of fatty acids are a predominant
Cholesterol

D1.3.8
Current Protocols in Food Analytical Chemistry
proportion of most foods. Cholesterol is usually brassicesterol, campesterol, 24-methylene-
a small proportion compared to triglycerides. cholesterol, and β-sitosterol. Because the rela-
In the HPLC analysis of cholesterol as intro- tive activities of cholesterol oxidase to stigma-
duced in the present protocol, however, residual sterol and β-sitosterol are 34% and 59%, re-
fatty acids in the cholesterol extract interfere spectively (while that to cholesterol is defined
with cholesterol detection and determination; as 100%), overestimation of cholesterol con-
fatty acids are co-eluted with cholesterol ben- tent in shellfish and its products usually occurs
zoate under the present HPLC conditions and during the enzymatic determination described
saturate the UV absorbance at 230 nm. Com- in Alternate Protocol 2. Therefore, although
plete removal of free fatty acids from the ether enzymatic determinations of cholesterol in
extract by sufficient saponification of the free food and foodstuffs other than shellfish should
fatty acids to corresponding potassium salts be reliable, those in shellfish may be misread.
avoids this problem (Newkirk and Sheppard, Several methodologies are known as effec-
1981). tive tools for the determination of cholesterol
Although sterols exist as cholesterol in most in food and foodstuffs, e.g., colorimetry using
food and food products including meat, fish, the Libermann-Burchard reaction (Naito and
fats and oils, and milk (Kushiro et al., 1980), David, 1984), Iatroscan thin-layer chromato-
certain shellfish contain six other analogs of graphy/flame ionization detection (Indrasena
sterol, including 22-trans-24-norcholesta- et al., 1991), enzymatic determination (Shen et
5,22-diene-3b-ol, 22-dehydrocholesterol, al., 1982), GC determination (Karkalas et al.,

Table D1.3.1 Sample Dilution for Enzymatic Measurement


of Cholesterol

Estimated amount of Ratio of sample to


Dilution factor
cholesterol per liter isopropanol (v/v)
<0.4 g None 1
0.4-4 g 1:9 10
4-40 g 1:99 100

Table D1.3.2 Troubleshooting

Problem Possible cause Solution


For GC
No peaks Decomposition of TMS ether Prepare derivative again
derivative due to moisture under dry conditions
Large tailing peak at solvent Interference of residual Evaporate pyridine under a
front pyridine in sample solution nitrogen stream
For HPLC
Unexpected large peak of Free fatty acid remaining in Add excess water and lower
cholesterol ether extract alcohol concentration during
ether extraction
Many unknown peaks Presence of oxidized Perform GC measurement as
cholesterol analogs recommended
For enzymatic determination
Underestimation of cholesterol Stabilizer in commercially Prepare fresh solution in the
available methanolic KOH laboratory
solution may inhibit
cholesterol oxidase
Too low optical density Too low concentration in Prepare sample: boil under
sample reflux with KOH, extract
with 1:1 ether/petroleum
ether, evaporate organic
phase, and dissolve residue
in isopropanol Lipid Composition

D1.3.9
Current Protocols in Food Analytical Chemistry
1982), HPLC determination (Osada et al., derivatization of cholesterol benzoate or for
1999), and HPLC/MS (Redden and Huang, TMS ether. The redistilled pyridine should be
1991). In general, enzymatic determination is kept in a desiccator stored in the dark until used.
superior to colorimetry to obtain true choles-
terol content. When food such as shellfish con- GC measurement
tains sterols other than cholesterol, the GC The TMS ether derivative should be kept in
determination is the most adequate method. a desiccator until analyzed because it is very
Although GC/MS also accomplishes good susceptible to moisture and hydrolyzes easily.
separation between and identification of all Excess amounts of pyridine usually result in a
sterol analogs, the instrument is too expensive large and tailing peak on a chromatogram and
to use for routine analyses of cholesterol. interfere with the baseline separation of the
cholesterol peak. It is therefore recommended
Critical Parameters to evaporate pyridine under a nitrogen stream
and to dissolve the residue in n-hexane. Excess
HPLC and GC measurement silylating agents usually produce a white resi-
Ethanolic potassium hydroxide irritates due in the resulting n-hexane solution. Micro-
skin. Laboratory glasses should be worn during filtration prior to GC injection is recom-
preparation to protect the eyes. mended.
When pyridine has a pale yellow color, it
should be distilled in glass prior to use for the

Table D1.3.3 Cholesterol Contents in Several Foods Determined by GC Measurementa

Cholesterol Cholesterol
Food Food
(mg/100 g sample) (mg/100 g sample)

Fish, roe, and shellfish: Fats and oils:


Salmon 73 Lard 109
Jack mackerel 71 Butter 210
Mackerel 70 Margarine 0-2
Sea bass 49 Head 123
Sea bream 82
Flounder 61 Dairy:
Tuna 46 Milk 11
Rainbow trout 70 Yogurt 11
Salmon, roe 400 Natural cheese 69-75
Herring, roe 261 Ice cream (low fat) 26
Cod, roe 295
Scallop 40 Meats:
Squid 312 Beef, rump 76
Prawn 175-228 Chicken, breast 131
Octopus 66 Pork, rump 84
Scallop 100 Mutton 93
Abalone 91 Turkey 72
Sea urchin 219 Duck 76
Rabbit 96
Eggs:
Whole egg 428 Others:
Egg yolk 1310 Mayonnaise 190
Dressing 145
aFrom 4th Revision of Table of Japanese Food Standard Ingredients, Resources Council, Science and Technology

Agency, Japan.

Cholesterol

D1.3.10
Current Protocols in Food Analytical Chemistry
Table D1.3.4 Comparison of Cholesterol Contents of Poultry Meat
and Cheese Determined by GC and Enzymatic Measurementsa

Cholesterol (mg/100 g sample)


Sample
GC method Enzymatic method
Chicken (raw):
Skin 128 130
Leg 90 91
Wing 98 97
Light meat 67 70
Dark meat 107 109
Chicken (cooked):
Skin 73 78
Leg 120 122
Wing 136 140
Light meat 80 80
Dark meat 92 93
Turkey (cooked):
Light meat 82 79
Dark meat 89 84
Cheese:
Edam 59 61
Double Gloucester 83 83
aValues are from Karkalas et al. (1982). Reprinted with permission from Blackwell Science

Ltd.

Table D1.3.5 Substrate Specificity of Cholesterol Oxidasea

Sterols Relative activity


Androsterone 0
Diosgenin 0
Testosterone 0
Estradiol 10
Stigmasterol 34
Dehydroepiandrosterone 37
β-Sitosterol 59
Cholesterol 100
β-Cholesterol 110
Pregnenolone 127
aValues are relative activity toward cholesterol and are from Kushiro et al. (1980).

Reprinted with permission.

Table D1.3.6 Comparison of Cholesterol Contents of Shellfish Determined by GC


and Enzymatic Measurementsa

Cholesterol (mg/100 g sample)


Sample
GC method Enzymatic method
Shijimi 125 147
Ark shell, Scapharca broughtonii 78 100
Short-neck clam, Ruditapes phillipinarum 76 95
Hard clam, Meretrix lusoria 69 89
Pacific oyster, Crassostrea gigas 76 107
aValues from Kushiro et al. (1980). Reprinted with permission.
Lipid Composition

D1.3.11
Current Protocols in Food Analytical Chemistry
HPLC measurement A typical HPLC chromatogram of choles-
Methanol used for the HPLC measurement terol benzoate obtained from dried egg yolk is
should be of HPLC grade with low absorbance shown in Figure D1.3.1.
at UV wavelengths below 230 nm. Dried-proc-
essed foods usually include a relatively large Time Considerations
amount of oxidized cholesterol analogs (Smith, For GLC and HPLC analysis, the extraction
1996). The benzoate derivatives of these oxides of total lipids from food requires overnight
do not separate from cholesterol benzoate by separation of the organic layer. If the sample
HPLC. Therefore, the GC measurement is more volume is small, however, centrifugation of the
suitable for the determination of cholesterol in extract may shorten the sample preparation
dried products with a high level of the oxidized time. Cholesterol derivatization requires <2 hr
cholesterol. depending on the number of samples. The peak
of TMS ether and cholesterol benzoate deriva-
Enzymatic measurement tives will be eluted within 10 min after the
The potassium hydroxide solution should be injection of the sample.
prepared fresh before use, because commer- Preparation of the sample for enzymatic
cially available methanolic potassium hydrox- measurement requires <30 min. One measure-
ide solution usually contains a stabilizer that ment requires <70 min.
may inhibit cholesterol oxidase. The amount of
cholesterol present in the test tube should be Literature Cited
between 8 and 160 µg under the presented AOAC (Association of Official Analytical Chem-
conditions to measure a sufficient difference in ists). 1990. 976.26 Cholesterol in multicompo-
nent foods. Gas chromatographic method. In
absorbance. The sample solution should be Official Methods of Analysis of the AOCS, 15th
diluted as shown in Table D1.3.1. ed. (K. Helrich, ed.) pp. 1103-1105. AOAC, Ar-
lington, Va.
Troubleshooting Carrol, R.M. and Rudel, L.L. 1981. Dietary fat and
Various problems and possible causes and cholesterol effects on lipoprotein cholesterol es-
solutions are shown in Table D1.3.2. ter formation via lecithin-cholestrol acyltrans-
ferase (LCAT) in vervet monkey. J. Lipid Res.
22:359-363.
Anticipated Results
United States regulations on nutrition label- Duncan, I.W., Culbreth, P.H., and Bartis, C.A. 1979.
Determination of free, total and esterified cho-
ing of foods require that cholesterol content be lesterol by high-performance liquid chromatog-
given and that it be analyzed by GC measure- raphy. J. Chromatogr. 162:281-292.
ment as shown in the AOAC method, which Indrasena, W.M., Paulson, A.T., Parrish, C.C., and
uses a packed column (Lewis et al., 1996; Ackman, R.G. 1991. A comparison of almina
AOAC, 1990). The reference value, which is a and silica gel Chromarods for the separation and
set of recommended nutrient intake levels of characterization of lipid classes by Iatroscan
cholesterol, is defined as 300 mg. However, the TLC/FID. J. Planar Chromatogr. 4:182-188.
Codex guideline does not request labeling of Karkalas, J., Donald, A.E., and Clegg, K.M. 1982.
cholesterol. Cholesterol contents in some foods Cholesterol content of poultry meat and cheese
determined by enzymic and gas-liquid chroma-
and foodstuffs determined by GC measurement tography methods. J. Food Technol. 17:281-283.
are summarized in Table D1.3.3.
Kushiro, H, Nakamoto, J., Fukui, I., Ogawa, Z.,
As shown in Table D1.3.4, there is good Yamaguchi, Y., Arisue, K., Hayashi, C., and
agreement between the cholesterol contents of Yamamura, Y. 1980. Cholesterol content in food.
poultry meat and cheese determined by GC and Rinnshou-eiyou 56:775-1980.
those measured by the enzymatic method. Lewis, C.J., Randell, A., and Scarbrough, F.E. 1996.
Cholesterol oxidase shows relatively wide Nutrition labelling of foods: Comparisons be-
substrate specificity to 3β-sterol analogs as tween US regulations and Codex guidelines.
shown in Table D1.3.5 As discussed above, Food Control 7:285-293.
cholesterol contents of certain shellfish, which Naito, H.K. and David, J.A. 1984. Laboratory con-
contain a relatively large amount of 3β-sterol siderations: Determination of cholesterol,
triglyceride, phospholipids, and other lipids in
analogs, may be unreliable when determined blood and tissues. Lab. Res. Methods Biol. Med.
by the enzymatic measurement as compared to 10:1-76.
results found using GC measurement, as shown Newkirk, D.R. and Sheppard, A.J. 1981. High pres-
in Table D1.3.6 (Kushiro et al., 1980). sure liquid chromatographic determination of
cholesterol in foods. J. Assoc. Off. Anal. Chem.
Cholesterol 64:54-57.

D1.3.12
Current Protocols in Food Analytical Chemistry
Osada, K., Ravandi, A., and Kuksis, A. 1999. Rapid Labovics, V.K., Antal, M., and Gaal, O. 1996. Enzy-
analysis of oxidized cholesterol derivatives by matic determination of cholesterol. J. Sci. Food
high-performance liquid chromatography com- Agric. 71:22-26.
bined with diode-array ultraviolet and evapora- By separation of cholesterol oxides from cholesterol
tive laser light-scattering detection. J. Am. Oil by TLC, it became possible to determine small
Chem. Soc. 76:863-871. amounts of cholesterol oxides by the enzymatic
Redden, P.R. and Huang, Y.-S. 1991. Automated method even in the presence of a quantity of choles-
separation and quantitation of lipid fractions by terol.
high-performance liquid chromatography and
mass detection. J. Chromatogr. 567:21-27. Lopez-Hernandez, J., Gonzales-Castro, M.J., and
Pineiro-Sotelo, M. 1999. Determination of ster-
Resources Council, Science and Technology ols in sea urchin gonads by high-performance
Agency. 1982. 4th Revision of Table of Japanese liquid chromatography with ultraviolet detec-
Food Standard Ingredients. Science and Tech- tion. J. Chromatogr. Sci. 37:237-239.
nology Agency, Japan.
Sterols in the sea urchin were separated by a C18
Shen, C.S.J., Chen, I.S., and Sheppard, A.J. 1982. column as a stationary phase with a UV detection
Enzymatic determination of cholesterol in egg at 205 nm. Three sterols (including desmosterol,
yolk. J. Assoc. Off. Anal. Chem. 65:1222-1224. fucosterol, and cholesterol) were identified and
Smith, L.L. 1996. Review of progress in sterol oxi- quantified.
dations: 1987-1995. Lipids 31:453-487.
Manzi, P., Panfili, G., and Pizzoferrato, L. 1996.
Normal and reversed-phase HPLC for more
Key References complete evaluation of tocopherols, retinols,
Bodzek, D., Bakowski, W., Wielkoszynski, T., carotenes and sterols in dairy products. Chroma-
Janoszka, B., Jaremczuk, B., Tarnawski, R., and tographia 43:89-93.
Typien, K. 1998. TLC and GC-MS determina-
tion of cholesterol in consumable fats. Acta A reversed-phase HPLC method was used for deter-
Chromatogr. 8:122-143. mination of cholesterol in dairy products. It evalu-
ated that a more complete evaluation of cholesterol
Full identification of isolated sterols from commer- contents in dairy products is necessary when the
cially consumable fats performed by GC/MS, and vegetables and fruits are present.
quantitative estimation of cholesterol content by
capillary GC with flame ionization detection. Nielsen, H. 2000. Application of chemical methods
to the determination of egg yolk. Lebens.Wiss.
Caboni, M.F. and Rodriguez-Estrada, M.T. 1997. Technol. 33:151-154.
High-performance liquid chromatography cou-
pled to evaporative light scattering detection in The adaptation of methods for lipid extraction and
lipid analysis: Some application. Seminars in quantification by colorimetric determination of
Food Analysis 2:159-169. either ester or cholesterol in egg yolk with some
contamination of egg white is described. Results are
An evaporative light scattering detector was cou- compared with those obtained by a conventional
pled with a UV spectrophotometer, and was applied enzymatic determination.
to HPLC for the quantitative determination of cho-
lesterol oxides in edible oils and fats. Pasin, G., Smith, G.M., and O’Mahony, M. 1998.
Rapid determination of total cholesterol in egg
Dutta, P.C., Caboni, M.F., Diczfalusy, U., Dionisi, yolk using commercial diagnostic cholesterol
F., and Dzeletovic, S. 1999. Measurements of reagent. Food Chem. 61:255-259.
cholesterol oxides in foods: Results of an inter-
laboratory comparison study. Spec. Publ. R. Soc. By using a commercial diagnostic cholesterol re-
Chem. 240:309-315. agent (enzymatic method) and gas chromatography,
the cholesterol contents of four different egg yolk
Study looking for good universal method for deter- preparations (including whole egg powder, fresh,
mination of eight analogs of cholesterol oxides in frozen, and dried egg yolk) were determined and
egg and milk powders. The results indicate difficulty compared.
in working with various food samples such as mixed
diets. Prygonski, K., Jelen, H., and Wasowicz, E. 2000.
Determination of cholesterol oxidation products
Fetouris, D.J., Botsoglou, N.A., Psomas, I.E., and in milk powder and infant formulas by gas chro-
Mantis, A.I. 1998. Rapid determination of cho- matography and mass spectrometry. Nahrung
lesterol in milk and milk products by direct 44:122-125.
saponification and capillary gas chromatogra-
phy. J. Dairy Sci. 81:2833-2840. A method for determination of cholesterol oxidation
products in milk powder and infant formulas is
For determination of cholesterol in milk and milk presented. Provides useful information on resolu-
products, samples are saponified without lipid ex- tion of cholesterol oxidizing products from unoxi-
traction in capped tubes with 0.5 M methanolic dized cholesterol on a GC capillary column.
potassium hydroxide solution by heating at 80°C.

Lipid Composition

D1.3.13
Current Protocols in Food Analytical Chemistry
Toivo, J., Piironen, V., Kalo, P., and Varo, P. 1998.
Gas chromatographic determination of major
sterols in edible oils and fats using solid-phase
extraction in sample preparation. Chroma-
tographia 48:745-750.
A capillary gas chromatographic method is de-
scribed for determination of major phytosterols and
cholesterol in edible oils and fats. To extract the
unsaponifiable matter and for sample cleanup,
solid-phase extraction with C18 absorbent was
used.

Contributed by Toshiaki Ohshima


Tokyo University of Fisheries
Tokyo, Japan

Cholesterol

D1.3.14
Current Protocols in Food Analytical Chemistry