Received: 23 June 2021 | Revised: 5 October 2021 | Accepted: 6 November 2021

DOI: 10.1111/jfpp.16146

ORIGINAL ARTICLE

Simultaneous determination of cholesterol, stigmasterol, and

β-­sitosterol contents in milk and dairy products

Lukáš Kolarič | Peter Šimko

Faculty of Chemical and Food Technology,

Institute of Food Science and Nutrition, Abstract
Slovak University of Technology in Since milk and dairy products are largely consumed food in the world, authentica-
Bratislava, Bratislava, Slovak Republic
tion is very important to control their real quality. Thus, the aim of this study was to
Correspondence develop and validate a rapid and reliable HPLC-­UV-­DAD method for the simultane-
Lukáš Kolarič, Slovak University of
Technology in Bratislava, Faculty of ous determination of cholesterol, stigmasterol, and β-­sitosterol contents in various
Chemical and Food Technology, Institute of dairy matrices (milk, cream, cheese, and butter). The validation showed great accu-
Food Science and Nutrition, Radlinského 9,
Bratislava 812 37, Slovak Republic. racy and precision of the developed method with the limit of detection of 2.32, 3.29,
Email: [email protected] and 0.99 µg/ml for cholesterol, stigmasterol, and β-­sitosterol, respectively. Except for
Funding information butter samples, the presence of phytosterols was not detected in other dairy samples
This publication was supported by at given analytical conditions. It can be concluded that the proposed HPLC method
the Operational program Integrated
Infrastructure within the project: Demand-­ is suitable for simultaneous determination of cholesterol and phytosterol contents in
driven research for the sustainable and milk and dairy products and it can serve as an authentication tool in food fat analysis.
innovative food, Drive4SIFood 313011V336,
co-­financed by the European Regional Novelty impact statement
Development Fund, and grant APVV-­ • The HPLC-­UV-­DAD method was validated on the determination of cholesterol
061-­2018.
and phytosterol contents in dairy products.
• The proposed method describes a rapid, reliable, and easy determination of dairy
adulteration and authentication as phytosterols should not be present in animal
food matrices.
• It was not confirmed the adulteration of 24 various samples (milk, cream, cheese,
and butter) except one butter sample.

1 | I NTRO D U C TI O N the similarities between the profiles of fatty acids in some vegetable
oils and milk (Sadeghi et al., 2018; Zarabadipour et al., 2020). Despite
Authentication is defined as the determination of the origin and purity that, the methods based on the analysis of fatty acid composition and
of foods based on their composition or genome, and it is an important physicochemical properties such as refractive index, melting point, or
matter on legal requirements, economic reasons, quality assurance, iodine value are often time-­consuming and involve several manual op-
or usage of safe products (Zarabadipour et al., 2020). As milk and erations (Khorsandmanesh et al., 2020). The determination of the ste-
dairy products have high nutritional value, global production as well rol composition is the most suitable method to differentiate vegetable
as consumption, it makes them a target of adulteration (Nascimento and animal fat as phytosterols, such as stigmasterol or β-­sitosterol, are
et al., 2017). Adulteration in dairy products is performed by removal not detectable in animal fat, or present only at trace levels, respec-
of some valuable substances, e.g., milk fat can be partially replaced tively (Kamm et al., 2002; Zarabadipour et al., 2020).
with low-­prized vegetable oils such as palm, sunflower, or soya bean Sterols are tetracyclic lipid components found in animals, plants,
oil (Sadeghi et al., 2018). The useful tool for dairy products authen- and microorganisms. Cholesterol is the major sterol of animal's fats,
tication could be thus the analysis of fatty acid composition or tria- while the most common sterols in plants are β-­sitosterol, campes-
cylglycerols (TAG); however, it has some drawbacks in the variations terol, and stigmasterol (Chen et al., 2015). The richest sources of
in animal feeding and different regional and seasonal conditions or phytosterols are vegetable oils and their derived products, cereal

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grains, cereal-­based products, and nuts (Saraiva et al., 2011). The samples (2 cow, 1 sheep, and 1 goat milk), 4 cream samples, 11
structure of phytosterols and cholesterol are similar to the differ- cheese samples, and 6 butter samples. In none of these, products
ence in double bound and carbon side chains (Sadeghi et al., 2018). was declared the addition of vegetable fats or oils.
The determination of sterols` content in dairy products is mainly
carried out for estimation of the total cholesterol content and detect-
ing the addition of vegetable fats (Poonia et al., 2016). According to 2.3 | Sample preparation
Poonia et al. (2016), several methods were developed for the detection
of vegetable or animal fats added to milk fats, e.g., a rapid gas chro- The sample preparation process was performed according to our
matography (GC) procedure, GC packed and short capillary columns, previous study (Kolarič & Šimko, 2020a). In the saponification, 5.0 g
GC long capillary columns, or the pyrogram fingerprints obtained by of milk samples, 1.0 g of cream and cheese samples, and 0.5 g of
pyrolysis. Capillary-­column GC in determining sitosterol or stigmas- butter samples were refluxed with 0.015 L of the methanolic solu-
terol in butter or concentrated butter is approved also as a reference tion of 1 mol/L KOH for 15 min. The unsaponified matter was ex-
method according to Commission Regulation (EC) no. 273/2008. In tracted with the mixture of n-­hexane and chloroform (1:1, vol/vol).
our previous article (Kolarič & Šimko, 2020a), it was shown a precise The total volume of solvent used for single extraction was 0.015 L.
and reliable method for the analysis of cholesterol content in butter For increasing the polarity of saponifiable residue, 10 ml of deion-
by high-­
performance liquid chromatography (HPLC). According to ized water was added. To avoid the formation of the emulsion dur-
Lagarda et al. (2006), the most used method for the determination of ing the extraction, 1 ml of 96% (vol/vol) ethanol was added to the
phytosterols in foods is based on GC with flame-­ionization detection saponified matter. The extraction process was duplicated. Then, the
(FID). However, the GC techniques often require laborious derivatiza- combined extracts were filtrated through anhydrous Na2SO 4 and
tion steps and high column temperatures. In comparison to GC and evaporated using a rotary vacuum evaporator (Witeg). The residue
spectrophotometric determination, the HPLC method was found to be was dissolved in 3 ml of methanol and the solution was filtered using
the most convenient and sensitive for the determination of cholesterol a syringe PTFE filter with 0.2 µm membrane (Agilent Technologies),
content (Kolarič & Šimko, 2020b; Osman & Chin, 2006). The sample and analyzed by HPLC.
preparation usually includes several steps, such as lipid extraction,
saponification, extraction of unsaponifiable matter to nonpolar sol-
vent, derivatization, and chromatographic detection (Borkovcová 2.4 | Chromatographic conditions
et al., 2009; Kolarič & Šimko, 2020a; Sadeghi et al., 2018). After the
isolation of the sterol fraction, all conjugated or esterified phytosterols The determination of cholesterol, stigmasterol, and β-­sitosterol con-
have to be converted into free molecules for GC analysis, which also tents was performed by HPLC analysis using an Agilent Technologies
extends the time of analysis (Inchingolo et al., 2014). 1260 infinity system equipped with a vacuum degasser, a quarterly
The objective of this study was to develop and validate a rapid pump, an autosampler, and the UV-­DAD detector, set at 205 nm.
and reliable HPLC method for the simultaneous determination of Isocratic elution was performed at a flow rate of 0.5 ml/min using
cholesterol, stigmasterol, and β-­sitosterol contents in milk and vari- the mobile phase consisted of acetonitrile/methanol 60:40 (vol/vol).
ous dairy products (cream, cheese, and butter) to serve as a possible The injection volume was 10 μl and the temperature was set at 30°C.
tool for the authentication of these foods. As a stationary phase, Zorbax Eclipse Plus C18 column (2.1 × 50 mm,
5 μm particle size, Agilent) was used with the guard column Zorbax
SB-­C18 (2.1 × 12.5 mm, 5 μm particle size, Agilent). The total run
2 | M ATE R I A L S A N D M E TH O DS time of analysis was 5 min with the retention time of cholesterol,
stigmasterol, and β-­sitosterol in 2.2, 2.5, and 2.9 min, respectively.
2.1 | Standards and reagents The analysis were recorded using the OpenLab CDS software,
ChemStationEdition for LC, and LC/MS systems (product version
Cholesterol, stigmasterol, and β-­
sitosterol standards were pur- A.01.08.108).
chased from Sigma-­Aldrich with a purity of ≥99%, 95%, and ≥90%,
respectively. Potassium hydroxide was purchased from Mikrochem,
chloroform, n-­hexane, ethanol, and sodium sulfate anhydrous from 2.5 | Method validation
Centralchem s.r.o., and methanol and acetonitrile (HPLC grade) from
VWR Chemicals (Fontenay-­sous-­Bois). The method validation was performed with the determination of
selectivity, linearity, the limit of detection (LOD), the limit of quan-
tification (LOQ), precision, and accuracy. The excellent selectivity
2.2 | Samples of HPLC analysis was confirmed by the comparison of the scanned
UV spectrum of the cholesterol, stigmasterol, and β-­sitosterol
Samples of milk products were randomly purchased in various standards with the chromatographic peaks of sterols eluted dur-
Slovakian supermarkets and consisted of 4 UHT pasteurized milk ing analysis. The linearity of the method was recorded from the
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KOLARIČ and ŠIMKO | 3 of 10

calibration curves, which were prepared by plotting the measuring TA B L E 1 The resolution parameters, linearity, LOD, and LOQ of
signal on the y-­a xis against the known quantity of the choles- the proposed HPLC-­UV method

terol, stigmasterol, and β-­sitosterol standards on the x-­a xis. 10 Cholesterol Stigmasterol β-­sitosterol
concentrations (2, 6, 10, 25, 40, 50, 75, 100, 300, and 350 µg/ml)
Concentration 2–­350 4–­250 4–­250
of standard cholesterol solution (1,000 mg/L), and 9 concentra- range [µg/ml]
tions (4, 5, 15, 25, 40, 50, 100, 150, and 250 µg/ml) of stigmasterol Slopea 10.8 ± 0.02 15.5 ± 0.1 8.9 ± 0.01
(500 mg/L) and β-­sitosterol (200 mg/L) standard solutions were
Intercept a 39.0 ± 2.8 32.0 ± 5.7 6.6 ± 1.0
injected to column. Each solution was analyzed in quadruple and
Correlation 0.99 0.99 1.00
average peak areas were calculated. The linearity was described by coefficient (r)
the correlation coefficient for linear regression (r). The LOD and
LOD [µg/ml] 2.32 3.29 0.99
LOQ were calculated as 3 or 10 times the standard deviation of
LOQ [µg/ml] 7.73 10.98 3.29
blanks divided by the slope of the calibration curve. The accuracy
Elution time (tR) 2.2 2.5 2.9
of the method was studied as a recovery test by a standard addi- [min]
tion method. The samples of milk, cream, and cheese were spiked
Capacity factor (K) 4.5 5.3 6.3
with cholesterol, stigmasterol, and β-­sitosterol standards in three
Separation factor (α) 1.2b 1.2c 1.4d
different quantities: 1,000, 1,500, and 2,000 µg for cholesterol,
Resolution factor 2.6e 3.1f 5.9g
and 25, 50, and 75 µg for stigmasterol and β-­sitosterol. The preci-
(Rs)
sion was determined by the repeatability and intermediate preci-
Abbreviations: LOD, limit of detection; LOQ, limit of quantification.
sion. The repeatability was recorded by injecting four replicates a
The values are expressed as mean ± standard deviation.
of samples in quadruple on the same day. The intermediate pre- b
Separation factor of cholesterol and stigmasterol.
cision was then evaluated on three different days. The precision c
Separation factor of stigmasterol and sitosterol.
was shown from the standard deviation (SD) and relative standard d
Separation factor of cholesterol and sitosterol.
deviation (RSD). The accuracy and precision of the proposed HPLC e
Resolution factor of cholesterol and stigmasterol in milk sample spiked
method for butter samples were described in our previous study with stigmasterol (50 µg).
f
(Kolarič, & Šimko, 2020a). Resolution factor of stigmasterol and sitosterol in milk sample spiked
with stigmasterol (50 µg) and β-­sitosterol (50 µg).
g
Resolution factor of cholesterol and β-­sitosterol in milk sample spiked
with sitosterol (50 µg).
2.6 | The resolution parameters of cholesterol,
stigmasterol, and β-­sitosterol
3 | R E S U LT S A N D D I S CU S S I O N
To clarify the resolution of cholesterol, stigmasterol, and β-­sitosterol
in milk samples, the following parameters were calculated: elution 3.1 | Method validation
time (tR), capacity factor (K), separation factor (α), and resolution fac-
tor (Rs) according to Ravisankar et al. (2019): The proposed HPLC method for the simultaneous determination of
( ) cholesterol, stigmasterol, and β-­sitosterol contents in milk and vari-
tR − t0
K= , (1) ous dairy products (cream, cheese, and butter) was firstly validated
t0
on the linearity, accuracy, and precision. It is important as many pre-
vious articles were focusing only on the method validation for cho-
K2
∝ = , (2) lesterol determination in milk but not phytosterols. In our previously
K1
published article (Kolarič & Šimko, 2020a), it was shown a complete
( ) in-­house validation of the method of the determination of choles-
2. tR2 − tR1
RS = . (3) terol content in butter and so in this study the validation parameters
wb1 + wb2
were made regarding milk, cream, and cheese samples.
The linearity of the method was recorded from the calibration
2.7 | Statistical analysis curves, and it was noticed that the method was linear over the range
of 2–­350 µg/ml for cholesterol, and 4–­250 µg/ml for stigmasterol
Results are expressed as mean ±standard deviation or as a percent- and β-­sitosterol. The linear correlation coefficients were at least
age. Regression and correlation analysis was performed using the 0.99, which approved excellent linearity. The regression param-
XLSTAT tool of Microsoft Excel 365 (version 2012, Microsoft). The eters of calibration curves are summarized in Table 1. From these
correlation analysis was done between the fat and cholesterol con- parameters, LOD and LOQ values for all monitored sterols were
tents of cheese samples. The validation parameters were at least calculated. It can be stated that both LOD and LOQ values for cho-
triplicated and the determination of cholesterol, stigmasterol, and lesterol, stigmasterol as well as β-­sitosterol were sufficient for the
β-­sitosterol duplicated. determination of their real contents in dairy products. According
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4 of 10 | KOLARIČ and ŠIMKO

to Albuquerque et al. (2016), the HPLC-­DAD method for the deter- TA B L E 2 The method accuracy (recovery)
mination of cholesterol content exhibits the LOD and LOQ values
Sample Spiked amount [µg] Recoverya [%]
on 3 and 11 µg/ml, which is similar to this study, but lower values
Cholesterol
were recorded in the UHPLC system, 0.7 and 2.4 µg/ml, respectively.
Milk 1,000 101.9 ± 1.9
As it is described by Borkovcová et al. (2009), LOD values for the
determination of cholesterol content in food samples are strongly 1,500 96.6 ± 5.6

connected to the analytical method as DLLME-­HPLC-­UV resulted in 2,000 98.2 ± 4.8

LOD of 0.01 µg/L, HPLC-­fluorimetric on 10 µg/L, or Electrophoresis Cream 1,000 99.0 ± 2.0
on 5 µg/L. It was also noticed that HPLC showed greater LOD and 1,500 100.2 ± 4.4
LOQ in comparison to GC or spectrometry (Kolarič & Šimko, 2020b; 2,000 99.1 ± 1.8
Osman & Chin, 2006). According to Borkovcová et al. (2009), the Cheese 1,000 102.0 ± 4.6
LOD values of the simultaneous determination of cholesterol and 1,500 100.7 ± 0.2
plant sterols in dairy products were varied from 5.2 to 14.7 mg/kg, 2,000 99.3 ± 4.4
which are higher values for stigmasterol and β-­sitosterol than esti-
Stigmasterol
mated in this research. Pokkanta et al. (2019) determined LOD and
Milk 25 103.2 ± 6.6
LOQ values for the HPLC-­DAD-­FLD determination of β-­sitosterol in
50 100.9 ± 2.7
rice bran and vegetable oil samples to 0.36 and 1.08 µg/ml, which
75 99.6 ± 3.2
is quite similar to these results, 0.99 and 3.29 µg/ml, respectively.
Cream 25 99.0 ± 3.5
Lower values of LOD and LOQ were reported by Ito et al. (2017),
for β-­sitosterol 0.29 and 0.98 µg/ml and for stigmasterol 0.37 and 50 96.8 ± 0.6

1.22 µg/ml, respectively; however, their method involves time 75 99.7 ± 3.9

and solvent consuming derivatization steps before fluorescence Cheese 25 97.1 ± 0.6
analysis. According to Sadeghi et al. (2018), the proposed GC-­FID 50 102.0 ± 0.5
method showed LOD and LOQ for stigmasterol analysis at 0.29 and 75 99.7 ± 2.9
0.88 µg/ml but higher values were reported for β-­sitosterol, 4.38 and β-­sitosterol
13.28 µg/ml, respectively. Milk 25 101.7 ± 5.0
The accuracy of the proposed method is shown in Table 2. The
50 98.6 ± 3.7
accuracy was expressed as a recovery by spiking samples with cho-
75 99.1 ± 3.1
lesterol, stigmasterol, and β-­sitosterol standards at three quantities:
Cream 25 99.2 ± 3.6
1,000, 1,500, and 2,000 µg for cholesterol, and 25, 50, and 75 µg for
50 98.2 ± 4.9
phytosterols. The recoveries were varied from 96.6% to 102.0% for
75 98.3 ± 0.2
cholesterol, 96.8 to 103.2 for stigmasterol, and 96.0 to 101.7% for
β-­sitosterol. Therefore, it was shown that the proposed HPLC-­UV Cheese 25 96.0 ± 3.4

method is suitable for the analysis of these sterols in milk products. 50 99.7 ± 0.5

A great recovery of the HPLC method for the analysis of choles- 75 98.1 ± 5.8
terol content in milk products was noticed also in other studies, e.g., a
The values are expressed as mean ± standard deviation.
Albuquerque et al. (2016) achieved a recovery of 111% in sour cream,
Bauer et al. (2014) described recoveries as varied from 100.63% to Ito et al. (2017) in the HPLC-­FLD analysis of phytosterols and choles-
103.90% in milk samples or Ahn et al. (2012) from 98.11% to 102.34% terol in plant foods. The average recovery for cholesterol, stigmas-
in milk containing emulsified foods. However, none of these articles terol, and β-­sitosterol was 91.2%, 87.5%, and 92.1%, respectively.
studied the simultaneous determination of cholesterol and phytos- The precision of the method was determined as the closeness
terols. In most studies, the analysis of phytosterols was made by GC of results between independent tests obtained under stipulated
after laborious pre-­treatment steps. Sadeghi et al. (2018) investi- conditions (IUPAC Technical Report, 2002). The results are shown
gated the recoveries of the GC-­FID method for the determination in Table 3. The RSD values of daily repeatability and intermediate
of cholesterol, β-­sitosterol, stigmasterol, and campesterol contents precision for cholesterol content in milk, cream, and cheese sam-
with values up to 101%. Du and Ahn (2002) also described a satis- ples were less than 5.0%, which is recommended for microconstit-
fying recovery of up to 99% in the simultaneous determination of uents analysis (Bauer et al., 2014). The described HPLC-­UV method
tocopherols, cholesterol, and phytosterols using GC. In this study, it thus showed great precision. The RSD of repeatability varied from
was thus noticed that the HPLC-­UV method is comparable to GC in 0.3% to 1.3% for milk, 0.8% to 2.3% for cream, and 1.2% to 4.0% for
the determination of sterols in foods regarding recovery. According cheese samples. This was also described in other studies showing
to Pokkanta et al. (2019), the HPLC-­UV-­FLD method showed a re- the determination of cholesterol content in milk products by HPLC
covery of β-­sitosterol at 98.6% and stigmasterol at 99.2% by their (Ahn et al., 2012; Albuquerque et al., 2016; Bauer et al., 2014). In our
analysis in rice bran samples. Lower recoveries were described by previous study (Kolarič & Šimko, 2020a), the RSD of repeatability
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TA B L E 3 The repeatability and intermediate precision of the proposed HPLC-­UV method

Milk Cream Cheese

a a
Mean content [mg/kg] RSD [%] Mean content [mg/kg] RSD [%] Mean contenta [mg/kg] RSD [%]

Repeatability
Day 1 104.43 ± 0.30 0.3 785.76 ± 5.86 0.8 769.58 ± 13.08 1.7
Day 2 105.95 ± 1.42 1.3 764.59 ± 17.38 2.3 787.88 ± 15.31 1.2
Day 3 111.81 ± 0.64 0.6 793.15 ± 17.03 2.2 750.12 ± 30.51 4.0
Intermediate 107.39 ± 3.19 3.0 781.17 ± 12.10 1.2 769.19 ± 15.42 2.0
precision

Abbreviation: RSD, relative standard deviation.

a
The values are expressed as mean ± standard deviation.

and intermediate precision in the determination of cholesterol con- 172.52 mg/kg, respectively. Neither stigmasterol nor β-­sitosterol
tent in butter samples ranged from 0.6% to 3.9% and 0.8% to 1.8%, was detected in milk samples. According to the results published
respectively. Higher RSD values for repeatability and inter-­day pre- by Ramalho et al. (2011), cholesterol mean values determined by
cision were observed by Saraiva et al. (2011) with 6.0% and 11.8% liquid chromatography in the commercial cow milk samples bought
for milk, respectively. in Portugal were 116 for whole samples, 64 for semi-­skimmed,
From the results of method validation, it can be thus concluded and 22 mg/kg for the skimmed samples. Manzi et al. (2013) de-
that the method has a suitable LOD, LOQ, and great accuracy and termined the average cholesterol content in Italian cow´s milk at
precision, which makes it sufficient for the simultaneous determi- 128 mg/kg. A higher average value of cholesterol content in goat´s
nation of cholesterol, stigmasterol, and β-­
sitosterol contents in milk (150.3 mg/kg) was also noticed by Borkovcová et al. (2009).
dairy products. This statement was also proved by the calculation Khorsandmanesh et al. (2020) analyzed sterol content in milk fat
of resolution parameters of these sterols in milk samples. Resolution by GC-­FID according to ISO method and found out that β-­sitosterol
factors for cholesterol, stigmasterol, and β-­sitosterol in spiked milk was detected in milk due to the animal feed while campesterol and
sample were higher than 1.7, which is desirable for rugged methods stigmasterol were not determined in pure milk fat. According to
(Ravisankar et al., 2019). Nurseitova et al. (2019), analysis of sterol content in commercial
milk samples bought in Kazakhstan revealed that almost 50% of
samples were falsified, especially low fat (<2.5%) UHT pasteurized
3.2 | The determination of cholesterol, milk samples with the β-­sitosterol content of 19.1% as a percent-
stigmasterol, and β-­sitosterol contents in various age of total sterol. Sadeghi et al. (2018) also found a small amount
milk products of stigmasterol and β-­sitosterol in Iranian milk samples, ranging
from 1.1 to 5.0 mg/kg and 14 to 53 mg/kg , respectively. According
The validated method was used for the analysis of cholesterol and to Zarabadipour et al. (2020), the sterol composition of milk con-
phytosterol contents in dairy products. As the major sterol in milk sisted of cholesterol (99.24%) and phytosterols (0.76%). The chro-
fat is cholesterol, the presence of stigmasterol or β-­sitosterol can matogram and 3D spectrum of the analysis of the milk sample
describe the adulteration with vegetable fat. The results are shown with the spiked stigmasterol (50 µg) and β-­sitosterol (50 µg) are
in Table 4. According to the article published by International Dairy shown in Figure 1. The average content of cholesterol in cream
Federation (IDF, 2008), whole milk should contain about 130 mg/L samples was 921.17 mg/kg with no detectable stigmasterol or β-­
of cholesterol and butter 240 to 280 mg/100 g, while other phytos- sitosterol amount. According to Han et al. (2007), the mean cho-
terols represent less than 1% of total sterols. The official standard lesterol content of the cream (36% fat content) was 1,370 mg/kg
method for the determination of sterol content in milk fat adopted and Piironen et al. (2002) determined that by GC on 769 mg/kg
by IDF is based on a gas liquid chromatographic analysis, directly (38% fat content).
on the unsaponifiable matter, without purification and derivatization
(ISO, 18252:2006, 2006).
3.2.2 | Cheese samples

3.2.1 | Milk and cream samples According to the report of the Food and Agriculture Organization of
the United Nations (FAO, 2019), world cheese exports increased up
The mean cholesterol content in cow´s milk samples (3.5% fat) to 0.8% in 2018, compared to 4.6% in 2017, which means that world
was 106.86 mg/kg, while higher values were found in sheep´s trade of cheese is still increasing. Despite that, Europe produced
milk sample (5.0% fat) and goat´s milk sample (3.0%), 190.80 and nearly 52% of the world´s cheese in 2013 (Manuelian et al., 2017).
 17454549, 2022, 1, Downloaded from https://ifst.onlinelibrary.wiley.com/doi/10.1111/jfpp.16146 by UNIVERSIDADE FEDERAL DE GOIAS, Wiley Online Library on [01/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
6 of 10 | KOLARIČ and ŠIMKO

TA B L E 4 The determination of cholesterol, stigmasterol, and β-­sitosterol contents in milk and dairy products

Declared fat content Cholesterol content Stigmasterol content β-­sitosterol

Sample [%] [mg/kg]b [mg/kg]b content [mg/kg]b

Milk samples
Cow milk 3.5 109.02 ± 6.04 ND ND
Cow milk 3.5 104.69 ± 0.05 ND ND
Sheep milk 5.0 190.80 ± 11.16 ND ND
Goat milk 3.0 172.52 ± 4.77 ND ND
Cream samples
1 33.0 934.47 ± 12.09 ND ND
2 30.0 855.78 ± 3.61 ND ND
3 30.0 965.52 ± 24.18 ND ND
4 30.0 928.91 ± 17.01 ND ND
Cheese samples
Mozzarella 40.0a 587.96 ± 17.08 ND ND
Feta 40.0a 548.18 ± 1.12 ND ND
Romadur 40.0a 582.21 ± 2.34 ND ND
a
Gouda 48.0 792.65 ± 16.83 ND ND
Gran Biraghi 30.0a 500.92 ± 35.58 ND ND
a
Grana Padano 32.0 814.76 ± 69.26 ND ND
Cheddar 48.0a 949.69 ± 15.37 ND ND
Parmigianno Reggiano 32.0a 1,034.74 ± 12.92 ND ND
Emmental 45.0a 799.10 ± 2.15 ND ND
Smoked cheese “Polooštiepok” 35.0a 589.74 ± 34.79 ND ND
Leerdammer 45.0a 769.36 ± 19.01 ND ND
Butter samples
1 82.0 2,124.01 ± 57.20 44.82 ± 2.03 22.72 ± 1.04
2 82.0 2,431.39 ± 92.18 34.80 ± 0.91 ND
3 82.0 2,399.28 ± 103.47 31.94 ± 2.21 ND
4 82.0 2,354.58 ± 27.43 34.02 ± 0.40 ND
5 84.0 2,406.99 ± 125.87 ND ND
6 82.0 2,760.00 ± 40.44 ND ND

Abbreviation: ND, not detectable.

a
Fat in dry matter.
b
The values are expressed as mean ± standard deviation.

The cholesterol content among the various cheese samples var- Grana Padano (814.76 mg/kg), German Emmental (799.10 mg/kg),
ied from 548.18 to 1,034.74 mg/kg. Stigmasterol and β-­sitosterol or Irish Cheddar (949.69 mg/kg), than soft cheeses, e.g., German
were not detected thus the adulteration of cheese samples was not Mozzarella (587.96 mg/kg) and Romadur (582.21 mg/kg) or Greek
proven. Cheese sterol composition may depend on several factors Feta (548.18 mg/kg). The same conclusions were reported by
such as the milk´s microbiological and chemical composition, the Manuelian et al. (2017). By the evaluation of cholesterol content of
cheese-­making technology, ripening time, and cheese factory condi- Greek cheese varieties, it was shown that the overall mean value was
tions (Manuelian et al., 2017). Cholesterol content of the USA cheese 776 mg/kg with the range from 390 to 1,152 mg/kg. Besides that,
varieties determined by capillary gas chromatography ranged from a strong correlation between cholesterol and fat content was found
809 to 1,248 mg/kg and similar values were reported for several for pooled data but the weaker correlation within individual cheese
European cheese samples, e.g., 890 mg/kg for Emmental or 715–­ varieties (Andrikopoulos et al., 2003). Kinik et al. (2005) estimated
778 mg/kg for Cheddar (Andrikopoulos et al., 2003). The choles- the cholesterol content of Turkish hard and soft cheeses between
terol content was more abundant in hard and semi-­hard cheeses, 464.7 and 1,389.9 mg/kg and also confirmed that the differences
e.g., Italian cheeses Parmigianno Reggiano (1,034.74 mg/kg) and may be explained by the various fat content of cheeses, origin, and
 17454549, 2022, 1, Downloaded from https://ifst.onlinelibrary.wiley.com/doi/10.1111/jfpp.16146 by UNIVERSIDADE FEDERAL DE GOIAS, Wiley Online Library on [01/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
KOLARIČ and ŠIMKO | 7 of 10

F I G U R E 1 The chromatogram and 3D spectrum at 205 nm of the analysis of milk sample with the spiked stigmasterol (50 µg) and β-­
sitosterol standard (50 µg)

production technologies. In this study, it was not found a significant the authentication analysis. The adulteration of butter can be de-
correlation between the fat and cholesterol contents of cheese sam- scribed in two periods: before and after the commercial manufac-
ples thus other factors probably influence the cholesterol content ture of margarine. Before the margarine era, cheaper substances,
such as the ripening stage or the type of the microbiological cul- such as chalk or potato starch, could be detected. However, after
ture media. According to Piironen et al. (2002), low-­fat cheese usu- the margarine invention, the addition of much cheaper margarine
ally contained 330 mg/kg, whereas cheese above 31% fat contained or vegetable oil was frequently applied (Deelstra et al., 2014).
770 mg/kg of cholesterol. The stigmasterol and β-­sitosterol contents In this study, the cholesterol content varied from 2,124.01 to
in the natural mozzarella cheeses varied from 0.0 to 0.49 mg/kg and 2,760.00 mg/kg. In most samples, the stigmasterol content was
0.59 to 1.05 mg/kg, respectively, which proves that the major sterol detected on average of 36.40 mg/kg but the values were below
in natural cheeses is cholesterol (Kim et al., 2014). LOQ. In sample no. 1, the β-­sitosterol content was also detected
(22.72 mg/kg). As this sample had the lowest cholesterol content,
and the highest stigmasterol and β-­sitosterol contents, it could
3.2.3 | Butter samples confirm the hypothesis that the sample was adulterated by vegeta-
ble oil. In Figure 2, the examples of chromatograms for butter sam-
Global butter exports expanded by 7.5% in 2018, which is one ple no. 5, probably adulterated butter sample no. 1, butter sample
of the highest increments in dairy products (FAO, 2019). Butter with a declared addition of vegetable oils, and pure margarine are
is a solid emulsion, mainly of the water-­in-­oil type, with the fat shown. According to them, the possibility of the authentication of
content minimum of 82%, the water maximum of 16%, while the butter samples is evident. According to Contarini et al. (2002), β-­
main sterol is cholesterol with no detectable other sterol's tracers sitosterol is the most abundant phytosterol in most vegetable fats.
(Commission regulation (EC) 2271/1999, 1999). Thus, the simulta- Similar results were noticed by Derewiaka et al. (2011), where the
neous determination of cholesterol, stigmasterol, and β-­sitosterol amount of β-­sitosterol in two samples was significantly higher and
contents in butter samples should be also a useful method for the cholesterol content significantly lower, so they also concluded
 17454549, 2022, 1, Downloaded from https://ifst.onlinelibrary.wiley.com/doi/10.1111/jfpp.16146 by UNIVERSIDADE FEDERAL DE GOIAS, Wiley Online Library on [01/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 of 10 | KOLARIČ and ŠIMKO

F I G U R E 2 The chromatograms at 205 nm of the analysis of butter samples: (a) butter sample no. 5, (b) probably adulterated butter
sample no. 1, (c) the example of butter sample enriched with vegetable oils, (d) the example of pure margarine with added vegetable oils
(sunflower, linseed, and rapeseed oil)

that these samples were adulterated. According to their study, the 4 | CO N C LU S I O N S

amount of cholesterol content in butter samples determined by
GC-­MS, which is also a reference method based on Commission In this study, the rapid and reliable HPLC-­U V-­DAD method for the
Regulation (EC) 2271/1999, varied from 176.8 to 262.8 mg/100 g. simultaneous determination of cholesterol, stigmasterol, and β-­
According to Borkovcová et al. (2009), the content of stigmasterol sitosterol contents for testing the authenticity of milk and various
and sitosterol in fresh butter was not detected, while according to dairy products was described. This method was validated on selec-
Sadeghi et al. (2018), a small amount of β-­sitosterol was detected tivity, linearity, accuracy, and precision. All validation parameters
in butter samples. The cholesterol content in butter is variable, showed suitable values according to the official requirements. As
e.g., 1,928–­2 ,263 mg/kg (Gonçalves & Baggio, 2012), 2,512.7–­ most of the previously published articles used GC with the time-­
3,690.4 mg/kg (Seçkin et al., 2005), and 2,043–­
3 ,824 mg/kg and solvent-­consuming derivatization steps for the determination
(Derewiaka et al., 2011). of phytosterols content, this method can serve as an alternative
 17454549, 2022, 1, Downloaded from https://ifst.onlinelibrary.wiley.com/doi/10.1111/jfpp.16146 by UNIVERSIDADE FEDERAL DE GOIAS, Wiley Online Library on [01/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
KOLARIČ and ŠIMKO | 9 of 10

for the easier authentication purposes of milk and dairy products No 1255/1999 as regards intervention on the market in butter and
cream. Official Journal of the European Union. https://eur-­lex.europa.
and thus support their nutritional and health aspects. Except for
eu/legal​-­conte ​nt/EN/TXT/PDF/uri=CELEX ​: 01999​R 2771​-­2 0070​
the butter samples, the stigmasterol and β-­sitosterol presence was 621&from=EN
not detected in milk and dairy products. However, in one butter Contarini, G., Povolo, M., Bonfitto, E., & Berardi, S. (2002). Quantitative
sample, the higher values of the phytosterols and lower values of analysis of sterols in dairy products: Experinces and remarks.
International Dairy Journal, 12, 573–­578. https://doi.org/10.1016/
cholesterol were observed; thus, the addition of vegetable fats to
S0958​-­6946(02)00050​-­X
the cream during the butter production was probably applied. Deelstra, H., Burns, D. T., & Walker, M. J. (2014). The adulteration of
food, lessons from the past, with reference to butter, margarine
C O N FL I C T O F I N T E R E S T S and fraud. European Food Research and Technology, 239(5), 725–­744.
The authors have declared no conflicts of interest for this article. https://doi.org/10.1007/s0021​7-­014-­2274-­0
Derewiaka, D., Sosińska, E., Obiedziński, M., Krogulec, A., & Czaplicki,
S. (2011). Determination of the adulteration of butter. European
AU T H O R C O N T R I B U T I O N S Journal of Lipid Science and Technology, 113, 1005–­1011. https://doi.
Lukáš Kolarič: Data curation; Investigation; Validation; Writing-­ org/10.1002/ejlt.20110​0 006
original draft. Peter Šimko: Conceptualization; Methodology; Du, M., & Ahn, D. U. (2002). Simultaneous analysis of tocopher-
ols, cholesterol, and phytosterols using gas chromatography.
Writing-­review & editing.
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j.1365-­2621.2002.tb087​0 8.x
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9EN/
Gonçalves, M. F. D., & Baggio, S. R. (2012). Evaluation of quality of butter
ORCID from different provenance. Food Science and Technology, 32, 629–­
Lukáš Kolarič https://orcid.org/0000-0003-1137-9717 635. https://doi.org/10.1590/S0101​-­20612​01200​5000091
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