Practicum 11

Cholesterol Analysis

Outline
Learning objectives 1
Basic Principle 1
Work Procedures 2
Materials 3
Case study 4
Questions 6

Learning objectives
1. Understand the principle of cholesterol analysis
2. Enable to calculate, analyze and interpret data of cholesterol analysis

Basic Principle
The importance of fat characterization is evident in many aspects of the food industry,
including ingredient technology, product development, quality assurance, product shelf life,
and regulatory aspects. The effort to reduce the amount of calories consumed as fat in the
USA accentuates the significance of understanding the lipid components of food. Lipids are
closely associated with health; the cholesterol or phytosterol compositions and amounts of
trans, saturated, and n−3/ω3 fatty acids are of great concern to consumers.

Many methods exist for the quantification of cholesterol and phytosterols in various matrices.
Consulting research literature will give an indication of current practice and methods that may
be less laborious or adapted for use with specific foodstuffs.

The lipid extracted from the food is saponified. The saponification process is a hydrolysis, with
acyl lipids being converted to water-soluble FFA salts. Other components (called the
unsaponifiable or nonsaponifiable matter) do not change in solubility after hydrolysis, and
thus they remain soluble in organic solvents. Cholesterol (in the unsaponifiable fraction) is
extracted and derivatized to form trimethylsilyl (TMS) ethers or acetate esters. This increases
their volatility and reduces problems of peak tailing during chromatography. Quantitation is
achieved using capillary GC.

GC quantitation of cholesterol is recommended since many spectrophometric methods are

not specific for cholesterol. In the past, samples such as eggs and shrimp have had their
cholesterol contents overestimated by relying on less specific colorimetric procedures. Other
GC, HPLC, and enzymatic methods are available. For example, cholesterol methods developed
for frozen foods and meat products eliminate the fat extraction step, directly saponifying the
sample; compared to the AOAC method outlined previously, they are more rapid and avoid
exposure to toxic solvents.

Cholesterol oxidation products as well as phytosterols can be quantified using the GC

procedure outlined for cholesterol. A wide range of methods for analysis of sterols exist in the
literature; most use TMS ether formation to increase volatility of the hydroxide containing
sterols and to improve chromatographic resolution (reduce peak tailing).

Cholesterol claims “free” is allowed if the product contains less than 2 mg per reference
amount and per labeled serving (or for meals and main dishes, less than 2 mg per labeled
serving). This is allowed if only when food contains 2 g or less saturated fat per reference
amount, or for meals and main dish products, per labeled serving size for “free” claims and
per 100 g for “low” and “reduced/less” claims must declare the amount of total fat next to
cholesterol claim when fat exceeds 13 g per reference amount of labeled serving (or per 50 g
of food if reference amount is small), or when the fat exceeds 19.5 g per labeled serving for
main dishes or 26 g for meal products For dietary supplements: cholesterol claims cannot be
made for products that are 40 Cal or less per serving

Work Procedures
AOAC Method 976.26 outlined here is representative of the various procedures available for
cholesterol determination. Lipids are extracted from the food, saponified, and the
unsaponifiable fraction is extracted. This is accomplished by filtering an aliquot of the
chloroform layer through anhydrous sodium sulfate and evaporating to dryness in a water
bath using a stream of nitrogen gas. Concentrated potassium hydroxide and ethanol are
added and the solution is refluxed. Aliquots of benzene and 1 N potassium hydroxide are
added and then shaken. The aqueous layer is removed, and the process is repeated with 0.5
N potassium hydroxide. After several washes with water, the benzene layer is dried with
anhydrous sodium sulfate, and an aliquot is evaporated to dryness on a rotary evaporator.
The residue is taken up in dimethylformamide. An aliquot of this sample is derivatized by
adding hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). Water (to react with
and inactivate excess reagent) and an internal standard in heptane are added, then the
solution is centrifuged. A portion of the heptane layer is injected into a GC equipped with a
nonpolar column. The HMDS and TMCS reagents are rapidly inactivated by water, and thus
the reaction conditions must remain anhydrous.
Materials
All reagents and standards were of analytical grade. Cholesterol standard (CAS: 57-88-5) was
from Sigma-Aldrich (Madrid, Spain) and had a purity ≥99%. Potassium hydroxide (KOH), n-
hexane, acetonitrile (ACN), dichloromethane (DCM), ethanol, methanol, and isopropanol (IPA)
were purchased from Merck (Darmstadt, Germany). Ultrapure water was prepared using a
Milli-Q filter system (Millipore, Bedford, MA, USA). Infant/Adult nutritional formula (SRM®
1849) and Fatty acids/cholesterol in frozen diet composite (SRM® 1544) were obtained from
National Institute of Standards and Technology (Gaithersburg, MD, USA).

Calibration curves were performed using seven standard concentrations. A stock solution of
cholesterol (1.25 mg/mL) was prepared by dissolving 25 mg of cholesterol into 20 mL of
ACN/IPA (70:30, v/v). The working standard solutions were prepared from the stock solution
by appropriate dilution to obtain the final concentrations of cholesterol (5, 70, 140, 200, 270,
330 and 400 µg/mL).

Samples were randomly collected from major supermarket chains. Sour cream, egg and egg
yolk were used for the validation of the methods. Four brands of frozen chicken nuggets (two
commercial brands and two supermarket brands in the pre-fried form); one sample of chicken
nuggets from a fast-food restaurant already cooked (baked) and two standard reference
materials were used for the application of the chosen validated method. The chicken nuggets
samples acquired in the pre-fried form were submitted to two processing methods: (i) deep
fried using a household fryer (180 ºC, 3-5 min), and (ii) baked in a 129 domestic oven (200 ºC,
15 min). All the samples were homogenized for 1 min at 5600 rpm using a high performance
homogenizer (Ultra Turrax® 131 , IKA, Staufen, Germany). Afterwards, samples were kept in
containers and stored in a deep freezer (at least -20 ºC). Each sample was analyzed in triplicate
and chromatographic analyses performed in triplicate. The cholesterol content of the
analysed samples are expressed as mg/100 g of edible portion on fresh weight basis.

In a 50 mL tube, 0.25 - 1 g of sample was weighed, 5 mL of ethanolic KOH (0.4 M, w/v) were
added and thoroughly mixed in a vortex for 1 min. Afterwards samples were heated in a water
bath at 50 ºC for 30 min. Then, the mixture was cooled at room temperature, 5 mL of ultrapure
water were added and 140 thoroughly mixed in a vortex. Cholesterol was extracted twice with
10 mL of n-hexane. An aliquot (3 mL) of the combined extracts was dried under nitrogen,
redissolved in 3 mL of mobile phase and aliquots analysed in both chromatographic systems.

An Alliance 2695 HPLC system (Waters, Milford, MA, USA), equipped with a Waters 2996 DAD
detector, using a SupelcosilTM LC-18-DB (150 x 4.6 mm I.D., 3.0 µm particle size) analytical
column protected with a SupelcosilTM LC-18-DB guard column (20 x 2.1 mm I.D., 5.0 µm
particle size), Supelco (Bellefonte, PA, USA) was used for separation and quantification of
cholesterol. The detection was achieved at 210 nm and the peak areas were quantified and
processed with 7 Empower™ version 2.0 software (Waters, Milford, MA, USA). The mobile
phase was ACN/IPA (70:30 v/v), which was filtered under vacuum through a 0.45 µm
(Millipore, Bedford, MA, USA) membrane filter and then degassed in an ultrasonic bath for 30
min. Column and auto-sampler temperatures were kept at 20 ºC, the flow-rate was 1.2
mL/min and total run time 8 min. A volume of 10 µL was injected into the chromatographic
system.

Case study
The ilustation video can be accessed via the following link:
Isolation and reaction of cholesterol https://www.youtube.com/watch?v=FSPKwcntS6Y
HPLC method https://www.youtube.com/watch?v=ZN7euA1fS4Y

Fig. 1. Optimization of the conditions for samples saponification. (A) Reaction time and (B)
Ethanolic KOH concentration.

Fig. 2. Chromatograms of cholesterol standard (0.40 mg/mL) using UHPLC chromatographic

system with different mobile phases. (1) ACN/DCM (95:5, v/v), (2) ACN/IPA (70:30, v/v), (3)
ACN/IPA (80:20, 476 v/v), (4) ACN/IPA (90:10, v/v), (5) ACN/IPA (95:5, v/v) and (6) ACN
(100%).
 Fig. 3. Chromatograms of cholesterol standard (0.14 mg/mL) using HPLC system (A) and
UHPLC system (B); and chromatograms from HPLC and UHPLC systems obtained respectively
for sour cream (C) 0.05 mg/mL and (D) 0.05 mg/mL; egg (E) 0.11 mg/mL and (F) 0.10 mg/mL;
and egg yolk (G) 0.19 mg/mL and (H) 0.16 mg/mL. λ= 210 nm.

Questions
1. Is cholesterol apart of mandatory components for Food Label Under Nutrition
Labeling and Education Act?
2. How much is Daily Reference Values of cholesterol?
3. What the meaning of the product contains cholesterol “nearest 5 mg increment”?
4. What the meaning that “palm fruit oil” contains zero cholesterol? How do you think?
5. What we can interpret a claim “free of cholesterol”?
6. Identify the key points in the procedure of cholesterol analysis that should need to be
considered to assure the data quality!
7. How to check the data quality?
8. According to the above result please check the data quality and interpret the result!
Which sample has the highest cholesterol level?