Journal of Chromatography B 1188 (2022) 123068

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Thin-layer chromatography in the authenticity testing of bee-products

Dušanka M. Milojković-Opsenica *, Jelena Ð. Trifković, Petar M. Ristivojević, Filip Lj. Andrić
University of Belgrade – Faculty of Chemistry, Studentski trg 12-16, 11158 Belgrade, Serbia

A R T I C L E I N F O A B S T R A C T

Keywords: Quality control, nutritional value and the monitoring of hazardous residues in honey bee- products have become
Thin-layer chromatography major topics for both producers and consumers. Due to its potential role in human health, bee-products rich in
Quality control bioactive compounds are becoming increasingly popular. This review aims to provide an overview of thin-layer
Authenticity testing
chromatography methods used in quality control, authenticity testing and chemical profiling of bee-products in
Honeybees’ products
Chemometrics
order to help scientists engaged in the field of bee-products chemistry to utilize the advantages of this technique
in the detection and elimination of fraudulent practices in bee-product manufacturing. Recently, hyphenation of
thin-layer chromatography, image analysis and chemometrics support bee-products ana­
lysis by simultaneous determination of analytes with different detection principles, identification of individual
bioactive compounds as well as structure elucidation of compounds. Highlighted opportunities of thin-layer
chromatography could encourage further investigations that would lead to improvements in the detection and
elimination of marketing fraudulent practices.

1. Introduction comprehensive information about unknown compounds or extending

the reliability of confirmation of known compounds. HPTLC could also
Natural and food products are complex mixtures composed of com­ serve as specific form of bioassay due to the possibility of derivatization
ponents of highly similar and/or dissimilar chemical structures which with free radicals (e.g., 2,2-diphenyl-1-picrylhydrazyl, DPPH), living
demand highly sophisticated techniques for reliable separation and cells (bacteria, yeasts, fungi), or enzymes. Careful choice of derivatizing
quantification [1,2]. agents in combination with chromatogram illumination under visible,
Chromatographic techniques such as high-performance thin-layer 254 nm or 366 nm ultraviolet light can enhance selectivity in signal
chromatography (HPTLC), high-performance liquid chromatography acquisition of target bands, offering information regarding analyte po­
(HPLC), and gas chromatography (GC), hyphenated with structure larity, spectral properties, presence or absence of some functional
elucidation techniques, proved to be reliable techniques for authenticity groups (based on the selective reaction of derivatization), metabolite
assessments. Each of them can be applied for the identification of a very pattern differences between samples, identification of major/minor
particular group of compounds, e.g. LC for proteins, amino acids, car­ band(s), or biological activity of individual compounds and their
bohydrates, vitamins, phenolic compounds, triglycerides, chiral com­ contribution to the total biological activity [6,7].
pounds, and pigments, while GC is more suitable for volatile or semi Authenticity assessments (regarding geographical and botanical or­
volatile compounds. However, a development of simple, fast, harmo­ igins) are mainly focused on fingerprint analysis, which could be defined
nized, and comprehensive analytical method, which could permit the as a set of characteristic chromatographic signals that could be used for
verification of compliance with the quality specification, is constantly sample recognition [8–10]. Namely, the entire HPTLC chromatogram is
encouraged [3]. Thanks to development of high-performance adsorbent treated as a unique multidimensional vector expressed as a line profile
layers and sophisticated instrumentation for sample application, chro­ from the starting point to the solvent front. Qualitative evaluation of
matogram development, derivatization and chromatogram evaluation, chromatograms is performed by image analysis. List of available soft­
HPTLC become a commonly used analytical techniques in quality con­ ware with highlighted advantages and drawbacks, as well as procedures
trol of natural, food and pharmaceutical products, along with HPLC and for HPTLC image analysis and multivariate treatment of fingerprints
GC [4,5]. Additionally, a vast number of chemical reactions can be obtained in food and natural products analysis, are already summarized
employed for derivatization (limited solely by the analyst), giving in a mini-review paper [11].

* Corresponding author at: University of Belgrade – Faculty of Chemistry, Studentski trg 12-16, P.O. Box 51, 11158 Belgrade, Serbia.
E-mail address: [email protected] (D.M. Milojković-Opsenica).

https://doi.org/10.1016/j.jchromb.2021.123068
Received 31 July 2021; Received in revised form 17 November 2021; Accepted 24 November 2021
Available online 27 November 2021
1570-0232/© 2021 Elsevier B.V. All rights reserved.
D.M. Milojković-Opsenica et al. Journal of Chromatography B 1188 (2022) 123068

Product authenticity with respect to description is mostly directed to [25,26], have been developed for the analysis of phenolic compounds in
quantitative determination of parameters specified in particular legis­ propolis samples. Concerning an application of HPTLC in testing of
latives. Densitometric data could be obtained with several devices such propolis, it is mainly directed to fingerprint analysis of phenolics, as
as mono-wavelength and diode-array scanners, charge-coupled device separation and evaluation is very difficult due to the complex chemical
(CCD) cameras, flatbed scanners, and fluorescence densitometry. Addi­ composition, and determination of authenticity regarding production
tionally, information extracted from HPTLC chromatograms, particu­ (botanical and geographical origin).
larly those obtained from fingerprint analysis, must be evaluated by Screening of complex phenolics mixtures of poplar type propolis
different unsupervised and supervised chemometric methods in order to performed by HPTLC indicate the existence of two or three main types of
efficiently extract the maximum useful information from the data [11]. European propolis, so-called orange (O-type), blue (B-type), and green
The aim of the present review is to provide a cross-section of the (G-type). Such grouping is based on the color of the band present after
HPTLC methods used for the determination of bee-products authen­ derivatization with polyethylene-glycol and 2-aminoethyl diphe­
ticity, one of the most frequently adulterated products, in order to nylborinate and illumination under UV light. The O-type of poplar
highlight the opportunities of this technique in quality assessments and propolis, apart from light blue and faint green bands, shows the presence
to encourage further investigations that would lead to improvements in of several strong orange bands, while B-type shows a pattern dominated
the detection and elimination of marketing fraudulent practices. by the blue color [27–29]. Phenolics, such as pinocembrin, galangin,
CAPE (caffeic acid phenethyl ester), chrysin, quercetin 3,7-di-O-methyl
2. Tracing bee-products authenticity ether, techtochrysin and pinostrobin are characteristic for O-type, while
p-coumaric acid, galangin 5-O-methyl ether and ellagic acid were
Bee-products are accepted as functional food, as they have a positive identified in B- and G-type, whereas caffeic acid, quercetin, and apigenin
impact on an individual’s health and physical performances, in addition appear in B- and O-type [30].
to their rich nutritional content and high bioactive components. They Development of the chromatographic profiles of propolis samples
could be used alone or could be added to other food products to increase collected from different geographic areas were based on application of
their nutritional value. They are rich in carbohydrates, proteins, essen­ HPTLC conditions optimized regarding the resolution of the analysed
tial amino acids and fatty acids, components that strengthen the im­ compounds in order to get chromatograms with highest number of sharp
munity and protect the overall body health. Due to the growing bands with maximum separation efficiency and lowest background
consumers’ interest for bee-products, their adulteration becomes noise (Table 1). Concerning the applied stationary phases, majority of
frequent. In order to protect the consumers, authorities such as Codex studies were performed on non-modified and RP-18 modified silica
Alimentarius standard, the European Honey Directive and other legis­ layers. Comparing chromatograms obtained after application of RP and
lations, regulate bee products authenticity at national level. However, NP systems on French propolis samples, Chasset and coauthors revealed
due to the growing global trade of honey and other bee-products and its that both modes of separations led to a similar pattern, but that the RP
health-promoting use, it is important to have international standards for method revealed a better differentiation between the phenolic com­
their authenticity [12]. The knowledge of the chemical composition of pounds, especially for caffeic acid, CAPE and galangin, and thus, a
bee-products is, therefore, of general interest in terms of their protec­ higher observable number of orange and blue fluorescent bands for both
tion. However, due to the fact that many factors might have an influence types of propolis [31]. Additionally, the RP separation mitigated satu­
on chemical composition, as well as structural variety of constituents, ration effects during the color channel analysis of the chromatogram
controlling the authenticity of bee-products is a hard task [13]. image prior to multivariate evaluation and provided better input data
The main bee-products that are used for human consumption are for the classification of propolis samples [31]. Only in one research the
honey, pollen, propolis, royal jelly and bee venom. This article will authors used amino modified layers, which belong to a group of hy­
summarize so far validated HPTLC methods used for authenticity testing drophilic modified silicas. It has extended range of selectivity, reduced
of honey, propolis and pollen, three the most important bee-products. surface polarity, and lessened the influence of the vapor phase on
retention behavior, and therefore better reproducibility compared to
3. Authenticity assessment of propolis hydrophilic non-modified silica and the nonpolar RP materials [10].
Among applied mobile phases, toluene (or n-hexane) / ethyl acetate /
Propolis is a natural resinous substance collected by honeybees (Apis formic acid (or acetic acid) mixed in different proportions were the most
mellifera L.) from different plant parts such as buds, branches, leaves and used developing solvents (Table 1). Some authors used mixtures of
exudates. Chemical composition of propolis is variable and depends dichloromethane / methanol (9:1, v/v), methanol / water (6:4, v/v),
upon the climate, season, geographic region, as well as local flora which chloroform / methanol / formic acid (44.1:3:2.35, v/v/v), or petroleum
are exploited by the bees. Significant differences might be found in ether / ethyl acetate (7:3, v/v). Jasprica et al. investigated the influence
propolis originated from various parts of the world, and today we could of eleven different mobile phases on TLC separation of the components
distinguish Euro-Asian (temperate, or poplar type), African, or green of Croatian propolis [32]. They used numerical taxonomic procedures to
Brazilian propolis, all of them with distinct chemical features and select the most efficient chromatographic systems which would enable
characteristic biomarkers [14,15]. Propolis is composed of resin and rational classification and selection of mobile phases. The most appro­
vegetable balsam (50%), wax (30%), essential and aromatic oils (10%), priate mobile phases for separation were chloroform / methanol /
pollen (5%), and other organic substances (vitamins, carbohydrates, (98–100%) formic acid (44.1:3:2.35, v/v/v), and n-hexane / ethyl ace­
minerals, amino acids) (5%). Resin contains mainly phenolics, including tate / glacial acetic acid (31:14:5, v/v/v).
flavonoids, phenolic acids, and their esters. Wide range of pharmaco­ Fingerprint analysis of propolis samples is oriented in two directions:
logical properties have been attributed to phenolic compounds from (a) visual inspections, and (b) comprehensive approach based on the full
propolis, mainly antibacterial, antiviral, antifungal, antiinflammatory, chemometric processing of collected data. Visual inspection of HPTLC
immunomodulatory and antioxidant [16]. The cross section of chemical chromatograms includes visual evaluation of chemical patterns between
composition, botanical origin and biological activity of poplar type samples, identification of unknown bands, fingerprinting of major and
propolis is given in the review [15]. To date, several analytical tech­ minor constituents based on band intensity, which is why it is subjective
niques such as ultraviolet–visible (UV–VIS) spectroscopy [16], cyclic and highly dependent on the analyst’s perception [32]. However, a high
voltammetry [17], electrophoresis [18], GC [19], HPLC [20], polarog­ level of confidence in quality control of propolis was obtained by
raphy [21], nuclear magnetic resonance (NMR) spectroscopy [22], combining HPTLC and chemometric analysis. HPTLC with selective
Fourier-transform infrared (FTIR) spectroscopy [23], near infra-red post-chromatographic derivatization provides information on polarity,
(NIR) spectroscopy [24] and different mass spectrometry techniques functional groups and spectral properties of marker compounds, while

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D.M. Milojković-Opsenica et al. Journal of Chromatography B 1188 (2022) 123068

Table 1
Application of TLC in analysis of propolis samples.
Sample description Target Chromatographic system Chemometric method Reference
compounds
Stationary phase Developing solvent

Serbian propolis Phenolics HPTLC glass plates (20 × 10 cm) Toluene-ethyl acetate-formic acid (6:5:1, v/v/v) PCA, HCA, PLSDA [10]
coated with NH2 modified silica gel
60 F254
Romanian propolis Phenolics HPTLC plates (20 × 10 cm) coated Toluene–ethyl acetate–formic acid (30:12:5,v/ Fuzzy hierarchical [27]
with silica gel 60 F254 v/v) clustering
German propolis Phenolics HPTLC glass plates (20 × 10 cm) n-Hexane – ethyl acetate – acetic acid PCA, HCA, LDA [28]
coated with silica gel 60 F254 (5:3:1, v/v/v)
German propolis Phenolics HPTLC plates (20 × 10 cm) coated n-Hexane – ethyl acetate – acetic acid [29]
with silica gel 60 F254 (5:3:1, v/v/v)
Egyptian propolis Phenolics HPTLC aluminium plates (20 × 10 Toluene: ethyl acetate: acetic acid (10: 4:0.25, v/ PLS-R, OPLS-DA, HCA [30]
cm) coated with silica gel 60 F254 v/v)
French propolis Phenolics HPTLC plates (20 × 10 cm) coated RP system: n-Hexane – toluene – ethyl acetate – PCA [31]
with silica gel 60 F254 formic acid– acetic acid (16:6:10:3:3, v/v/v/v/
HPTLC plates (20 × 10 cm) coated v),
with RP-18 modified silica gel 60 NP system:
F254 n-Hexane – ethyl acetate – acetic acid (5:3:1, v/
v/v)
Croatian propolis Phenolics HPTLC glass plates (20 × 10 cm) Chloroform-methanol-(98–100%) formic acid [32]
coated with silica gel 60 F254 (44.1:3:2.35, v/v/v)
Serbian, Croatian and Phenolics HPTLC glass plates (20 × 10 cm) Toluene–ethyl acetate– PCA [33]
Slovenian propolis coated with silica gel 60 F254 formic acid (6:5:1, v/v/v)
Turkish propolis Phenolics HPTLC glass plates (20 × 10 cm) n-Hexane – ethyl acetate – acetic acid (5:3:1, v/ PCA [34]
coated with silica gel 60 F254 v/v)
Anatolian Propolis Phenolics HPTLC glass plates (20 × 10 cm) n-Hexane, ethyl acetate, glacial acetic acid PCA [35]
coated with silica gel 60 F254 (5:3:1, v/v/v)
Chinese propolis Phenolics HPTLC glass plates (20 × 10 cm) Toluene-ethyl acetate-formic acid (10:4:0.5, v/ PCA, HCA, k-means [36]
coated with silica gel 60 F254 v/v) clustering, ANN and SVM
Greece propolis Phenolics HPTLC glass plates (20 × 10 cm) Dichloromethane-methanol (90:10, v/v) PCA [37]
coated with silica gel 60 F254
Turkish propolis Phenolics HPTLC glass plates (20 × 10 cm) Petroleum ether-ethyl acetate (7:3, v/v) [38]
coated with silica gel 60 F254
Thai stingless bee Phenolics HPTLC glass plates (20 × 10 cm) Toluene-ethylacetate-formic acid (8:2:0.1, v/v/ [39]
propolis coated with silica gel 60 F254 v)
Serbian propolis Phenolics HPTLC glass plates (20 × 10 cm) Toluene–ethyl acetate– PLS [40]
coated with silica gel 60 F254 formic acid (6 : 5 : 1, v/v/v)
Propolis from South Phenolics HPTLC glass plates (20 × 10 cm) Methanol-water (60:40, v/v) [41]
Africa coated with silica gel 60 F254

chemometrics provides identification of botanical/geographical neural network (ANN), support-vector machine (SVM), linear discrimi­
markers, information about similarity/dissimilarity as well as prediction nant analysis (LDA), partial least square - discriminant analysis (PLS-
of classification of unknown samples [11]. Proper qualitative evaluation DA), partial least squares regression (PLSR).
of chromatograms include image analysis used for conversion of HPTLC Several articles reported a combination of HPTLC with other tech­
tracks in line profiles or/and numerical data sets. Several freely avail­ niques for further potential improvement with regard to differentiation.
able, user-friendly software packages for image evaluation can provide Shown on the example of French propolis samples, a strategy for an
means for simple and fast image processing, and possibility for data improved quality control in which HPTLC fingerprints were evaluated in
evaluation of image. However, before multivariate data analysis, raw combination with selected mass signals obtained by desorption-based
data obtained after image processing and signal acquisition, must be scanning mass spectrometry (MS), was demonstrated. After HPTLC-
subjected to correction which include denoising, baseline removal, image analysis-chemometric evaluation, characteristic phenolic
target peak alignment, and normalization. List of applied supervised and markers for differentiation of the propolis types were further discovered
unsupervised chemometric methods in evaluation of HPTLC data is after scanning direct analysis in real time (DART)-MS. HPTLC-DART-MS
presented in Table 1. was selected due to its ease of operation, i.e. a simple scan along the
Significance of the chemometric approach in evaluation of HPTLC chromatogram track using a substantially modified DART interface.
data in quality assessments study of propolis is confirmed in numerous Authors concluded that HPTLC image analysis combined and expanded
articles. Propolis samples from Romania were classified on two types by mass spectrometric analysis permitted a fast multivariate analysis of
according to their geographical origin and local flora; samples from the propolis samples [31]. Shown on the example of German propolis
areas with meadows contain orange bands, together with blue and green samples, a high level of confidence was obtained when combining
ones, while in samples originating from forest region orange zones were orthogonal approaches (HPTLC and DART-MS) for sample character­
not present. Additionally, three subgroups within the first group were ization. HPTLC with selective post-chromatographic derivatization
found according to the spreading and variety of the meadow, while provided information on polarity, functional groups and spectral prop­
within the second group two subgroups were identified according to the erties of marker compounds, while information on possible elemental
dominant type of the forest, deciduous or resinous (for applied multi­ formulae of principal components (phenolic markers) was obtained by
variate methods see Table 1) [27]. The existence of three main types of DART-MS [28]. Egyptian propolis samples were analysed by HPTLC-
European poplar propolis (O-, B- and G-type) was confirmed in samples qualitative untargeted method and specific markers were further eval­
from Serbia, Croatia and Slovenia [10,32,33], Turkey [34,35] (Fig. 1-I), uated by validated HPTLC-quantitative target method. Observed trends
China [36], Egypt [30] and France [31], after the application of che­ among data were fulfilled with the results of NIR spectroscopy. Authors
mometric methods such as principal component analysis (PCA) (Fig. 1- concluded that precise discrimination and high-quality assessment of
II), hierarchical cluster analysis (HCA), k-means clustering, artificial chemical and effective consistency of propolis samples could be

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D.M. Milojković-Opsenica et al. Journal of Chromatography B 1188 (2022) 123068

quantitation of α- and γ-mangostins in mangosteen propolis and iden­

tified the extraction solvent and bee species affording the best yield of
these constituents.
HPTLC coupled with bioactivity-based detection by bacteria cells or
DPPH radicals provide high-throughput, simple and low-cost bio­
profiling. Bioautography represents preliminary bioactivity screening of
natural products. Several HPTLC-bioautography methods have been
reported for elucidating antimicrobial, antifungal, and antioxidante
inhibiting compounds of propolis. This technique was applied for
determination of antimicrobial activity of poplar type propolis from
Serbia [40]. Orange type of propolis exhibited higher antimicrobial
activity compared to the B-type. PLS modelling was performed on the
HPTLC data set and the resulting models might qualitatively indicate
that caffeic acid, quercetin, naringenin, and chrysin, play an important
role in the activity exhibited by the propolis samples. Using South Af­
rican propolis as an example, the application of HPTLC-bioautography
in tandem with mass spectrometry was investigated for the rapid iden­
tification of antimicrobial and anti-quorum sensing (anti-QS) com­
pounds [41]. Antifungal activity of the propolis against Candida albicans
is attributed to pinocembrin, while pinobanksin, one unidentified
compound and pinobanksin 3-O-pentanoate or 2-methylbutyrate were
found to be active against all of the evaluated Gram-positive and Gram-
negative bacteria.
HPTLC coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) detec­
tion is a handy technique for screening of antioxidant capacity of each
separated component in a mixture or extract. It is cost- and time-
effective, it is able to test a high number of samples simultaneously,
including the reference compounds, it provides easier comparison of the
activity profiles of different samples and generalizes any possible ac­
tivity contribution of other components in the sample. Contribution of
each antioxidant components in Turkish propolis samples, was analysed
using HPTLC-DPPH˙ assay [34,35]. Results showed that O-type of
propolis exerted higher antioxidant activity than the other propolis
types. Moreover, quercetin, caffeic acid, caffeic acid phenyl ester,
pinobanksin and galangin had significant contributions to the antioxi­
dant activity of propolis.

4. Authenticity assessment of honey

Honey is a natural, sweet, and complex substance produced by honey

bees (Apis melifera L.). Due to its specific taste, nutritional and phar­
maceutical value, honey is commonly used in the food industry, medi­
cine, as well as manufacturing of cosmetic products [42]. Honey is a
concentrated aqueous solution of different carbohydrates, including
fructose, glucose, maltose, sucrose and other oligo- and polysaccharides.
It also contains a complex mixture of amino and organic acids, minerals,
Fig. 1. I) HPTLC chromatograms of (a) hydroalcoholic propolis extracts at 366
aroma compounds, flavonoids, vitamins, pigments, waxes, pollen grains,
nm, developing solvent system: n-hexane-ethyl acetate-acetic acid (5:3:1, v/v/
v), derivatization: NP/PEG 400; (b) hydroalcoholic propolis extracts at white
several enzymes and other phytochemicals [43]. The composition and
light, developing solvent system: n-hexane-ethyl acetate-acetic acid (5:3:1, v/v/ properties of this complex mixture depends on different factors such as
v), derivatization: DPPH˙ solution; propolis chloroform extracts at (c) 366 nm bee species, nectar-providing plant species, geographical area, season,
and (d) white light, developing solvent system: toluene-ethyl acetate (95:5, v/ mode of storage, and harvest technology and conditions [44,45]. To
v/v), derivatization: Anisaldehyde reagent; II) Principal component analysis to date, analytical methods such as HPLC, GC–MS, spectroscopic methods
determine botanical origin of Turkish propolis: a,b) mutual projections of factor such as NMR, FTIR and NIR have been used for metabolomic studies of
scores, c) loadings for the PC1, d) loadings for the PC2. Figures were taken from honey [46–52]. Also, other analytical methods such as capillary elec­
the G E. Guzelmeric et al. [34], Copyright licence no: 5181990490729. trophoresis, amperometric methods, ion chromatography as well as
immunoassays have found application in quality control of honey
obtained by combining HPTLC with NIR spectroscopy [30]. Chemical [53–56]. HPTLC has not been used extensively in honey authentication.
profiling of Greek propolis samples was investigated using HPTLC and Only a few articles have been published dealing with application of
1
H NMR spectroscopy [37], while fingerprint analysis of Turkish prop­ HPTLC in determination of honey authenticity with respect to descrip­
olis was performed by comparative application of TLC and GC–MS [38]. tion (natural, organic, raw, or unheated), while authenticity in regard to
Combining two techniques, more information on samples in the light of production (botanical or geographical origin) were started to evaluate
their authenticity were obtained. recently, from 2017 onwards. Concerning an application of HPTLC in
Chewchinda and Vongsak proposed a method for the quality testing of honey, it is mainly directed to quality assessments of carbo­
assessment of propolis in order to ensure the efficacy and safety of hydrates, and determination of botanical origin according to phenolic
related raw materials and nutraceutical products [39]. They developed a profile and lipophilic compounds.
simple, fast, accurate, and precise HPTLC method for the simultaneous Due to the constant increase of consumer interest in natural and

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D.M. Milojković-Opsenica et al. Journal of Chromatography B 1188 (2022) 123068

healthy food products such as honey, it is frequently subjected to botanical sources in the foraging area of the beehive, absolutely pure
adulteration either by direct addition of inexpensive and artificial unifloral honeys are very rare. Therefore, botanical origin of natural
sweeteners, sucrose syrups that are produced from sugar beet, high products, as well as honey, are mainly determined based on the profile of
fructose corn syrup (HFCS), maltose syrup, or by adding industrial sugar secondary metabolites, such as phenolics. However, in the case of honey,
(glucose and fructose) syrups obtained from starch, or by feeding the bee determination and identification of such compounds is challenging due
colonies excessively with these syrups during the main nectar period to their low concentrations, which further complicates their isolation
[12]. Detection of adulterants added to honey by HPTLC is limited due and choice of analytical method applied for their identification [59].
to the hydrolysis of oligosaccharides and polysaccharides and therefore This might be a reason for the limited application of HPTLC in the
presence of false positive components, particularly smaller oligosac­ authenticity assessment of honey.
charides (DP 3–6) in pure honeys. Puscas et al. validated a new HPTLC Chromatographic systems used for determination of honey authen­
method for detecting the adulteration in honey based on fructose/ ticity with respect to production were optimized regarding the resolu­
glucose ratio and sucrose content [57]. The applicability of the method tion of the analysed compounds, separately for each observed botanical
was checked by analyzing the glucose, fructose and sucrose in origin (Table 2). Similar to conditions applied for HPTLC analysis of
commercially available honeys. The adulteration was confirmed in propolis, the most oftenly used mobile phases were toluene (or chloro­
several honey samples with fructose and industrial sucrose. Addition­ form) / ethyl acetate / formic acid (or acetic acid) in different pro­
ally, they marked acacia honey as a more frequently adulterated honey portions, and separation was performed on silica gel. Additionally, a
type, probably due to its mild taste that the fraudulent producers prefer comprehensive TLC approach for determination of botanical origin de­
to raise by fructose addition. Detection of adulteration, the identification mands the full chemometric processing of collected data (Table 2).
of syrup adulterants, as well as an estimation of the level of adulteration, The utility of HPTLC determination of phenolic constituents for the
could be achieved by recently validated HPTLC method for quantifica­ authentication of honeys was demonstrated in several studies [59–63].
tion of glucose, fructose, sucrose and maltose complemented by the Additionally, HPTLC was applied in the evaluation of non-sugar con­
HPTLC fingerprinting of an organic sample extract [58]. The authors stituents [64,65], and lipophilic fraction of honey [66]. The developed
demonstrated that for common adulterants like glucose, fructose, brown procedures facilitate differentiation of varietal honeys (acacia, lime,
rice, corn starch, maltose, treacle or maple syrups, which contain only phacelia, goldenrod, pine, chestnut, sunflower, willow, buckwheat,
one, two or a combination of common sugars, HPTLC sugar profiling heather, manuka honey, Jarrah (Eucalyptus marginata) and Marri (Cor­
alone may be sufficient to detect the adulteration due to the presence of ymbia calophylla) honeys). Evaluations were performed by fingerprint
noticeable quantities of sucrose and/or maltose (Fig. 2). They noted that analysis or by quantification of specific compounds. Fingerprint analysis
even if the adulterants consist of only glucose and fructose that are was used to determine the characteristic patterns, band profiles, of
added to imitate the fructose to glucose ratios of naturally occurring investigated honey types. In these studies visual examination prevails
honeys, their deliberate addition to the honey can be detected via over the chemometric approach. The authors concluded that HPTLC
reduced peak intensities in the HPTLC profile of the organic extract. analysis could become a very useful rapid quality control instrument. A
Determination of botanical origin of honey is a hard task due to the database of reference honey HPTLC profiles which would include
fact that the floral source may be exactly determined only if the honey marker compounds could be a useful tool in the detection of honey
originates completely or mostly from a unique source and if its sensory, adulteration. Honey authenticity studies enable a more complete
physicochemical, and microscopic characteristics correspond to that assessment of its quality and the identification of emerging threats to
source. However, as honeybees collect nectar or honeydew from several honey quality.

Fig. 2. Images taken at T White light; (a) Track 1—Manuka honey, Track 2—Manuka honey + Glucose 30%, Track 3—Manuka honey + Maple syrup 30%; (b) their
respective chromatograms. Figure was taken from the M.K. Islam et al. [58], Copyright licence: Open access.

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D.M. Milojković-Opsenica et al. Journal of Chromatography B 1188 (2022) 123068

Table 2
Application of TLC in analysis of honey samples.
Sample description Target Chromatographic system Derivatization/visualization/ Reference
compounds detection/chemometric method
Stationary phase Developing solvent

Lime, Polyfloral, acacia Carbohydrates HPTLC aluminium plates Ethyl acetate–pyridine–water–acetic Derivatization with diphenylamine [57]
honey (20 × 10 cm) coated with acid (6:3:1:0.5, v/v/v/v) and aniline hydrochlorid
silica gel 60 F254 Visualization was performed under
visible light.
Manuka, Jarrah, Marri, Carbohydrates TLC glass plates (20 × 1-butanol: 2-propanol: aqueous boric Derivatization with aniline- [58]
Karri, Peppermint and 10 cm) coated with silica acid (5 mg/mL) (30:50:10, v/v/v) diphenylamine-phosphoric acid
White Gumhoney gel 60 F254 reagent.
Visualization was performed under
white light.
Phacelia honeys Phenolics TLC glass plates (20 × Chloroform-ethyl Visualization was performed under [59]
10 cm) coated with silica acetate-formic acid (5:4:1, v/v/v) white light and 366 nm or by spraying
gel 60 F254 with 1% methanolic AlCl3
Acacia and lime honey Phenolics HPTLC plates (20 × 10 Chloroform-ethyl acetate-formic acid Visualization was performed under UV [60]
cm) coated with silica gel (5:4:1, v/v/v) light at 254 nm and at 366 nm or by
60 F254 spraying with 1% methanolic AlCl3.
PCA
Willow, buckwheat, heather, Phenolics TLC glass plates (20 × Chloroform-ethyl acetate-formic acid Visualization was performed under UV [61]
pine honeydew, and 10 cm) coated with silica (5:4:1, v/v/v) light at 254 nm and at 366 nm or by
manuka honey gel 60 F254 spraying with 1% methanolic AlCl3.
PCA, HCA
Goldenrod honey Phenolics HPTLC aluminium plates Toluene–ethyl acetate (95:5, v/v) Derivatisation with an anisaldehyde [62]
(20 × 10 cm) coated with reagent.
silica gel 60 F254 Visualization was performed under
white light and 366 nm
Commercialy available Phenolics TLC glass plates (20 × Two different developing solvent Derivatization with NP reagent [63]
samples of honey 10 cm) coated with silica mixtures: (i) followed by PEG 400.
gel 60 F254 developing system A: ethyl Visualization was performed under
acetatedichloromethane- 366 nm
glacial acetic acid-formic acid–water
(100:25:10:10:11, v/v/v/v/v); (ii)
developing system B:
n-hexane–ethyl acetate-glacial acetic
acid (5:3:1,
v/v/v).
Jarrah, Marri honeys Non-sugar HPTLC glass plates (20 Toluene-ethyl acetate-formic acid (6:5:1, Derivatization with vanillin. [64]
constituents × 10 cm) coated with v/v) Visualization was performed under
silica gel 60 F254 white light, 254 nm and at 366 nm
Jarrah, Marri honeys Non-sugar HPTLC glass plates (20 Toluene-ethyl acetate-formic acid (6:5:1, Derivatization with vanillin. [65]
constituents × 10 cm) coated with v/v) Visualization was performed under
silica gel 60 F254 white light and 366 nm
Rape, buckwheat, clover, Lipophilic HPTLC aluminium plates Toluene-ethyl acetate (80:20, v/v) Derivatisation with [66]
willow, milk thistle, fractions (20 × 10 cm) coated with an anisaldehyde reagent.
dandelion, raspberry and silica gel 60 F254 Visualization was performed under
sweet yellow clover honey white light and at 366 nm.
PCA
Manuka, Coastal Antioxidante HPTLC glass plates (20 Toluene: ethyl acetate: formic acid Derivatization with DPPH reagent y[67]
Peppermint, Marri, Karri, compounds × 10 cm) coated with (6:5:1, v/v/v)
River Red Gum, silica gel 60 F254
Multifloral honey

In addition to determining the botanical origin of honey, evaluation source of nutrients (lipids, proteins, amino acids, saccharides, phenolics,
of the antioxidant activity, which is directly related with its bioactive vitamins, and minerals) for bee larvae and other members of a bee
compounds, was performed [63,67]. Antioxidant HPTLC-DPPH finger­ colonies. Due to its numerous health promoting effects, bee pollen is
printing method facilitates the visualization of individual constituents considered as a valuable natural product and potential food supplement.
that contribute to the overall antioxidant activity of the honey, even if However, its use as a food supplement depends on its chemical
they are not yet chemically identified. Additionally, the method allows composition, and consequently from botanical and geographical origin.
comparative analysis of the antioxidant fingerprints of honeys of However, even monofloral bee pollen exhibits huge variation in chem­
different botanical origin and indicates differences in their individual ical constituents. Although, in such cases 45% of pollen grains originate
bioactive constituents. Islam et al. indicated that HPTLC-DPPH method from one particular plant species, and therefore guarantees particular
enables the tracking of changes in antioxidant activity of individual botanical uniqueness, still the 55% of grains that come from the mixture
honey constituents over time upon exposure to different temperature of plants can significantly alter the chemical composition, and biological
conditions, which demonstrates the potential value of the method for in- activity. Moreover, chemical composition can be strongly influenced by
process quality control [67]. floral microsorounding at the beehive spot. Although palynological
analysis can provide insights into botanical constituents of bee pollen,
5. Authenticity assessment of pollen and consequently can be used to trace its botanical origin, it does not
give any information regarding biological activity and quality. There­
Bee pollen is produced by the agglutination of pollen grains, nectar fore, determination of chemical composition of bee pollen is of the
and salivary secretions of bees. Salivary secretions promote further utmost importance. However, only few reports are published with re­
enzymatic transformations of phytochemicals [68,69]. It is the main gard to application of TLC in bee pollen analysis (Table 3).

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D.M. Milojković-Opsenica et al. Journal of Chromatography B 1188 (2022) 123068

Table 3
Application of TLC in analysis of pollen samples.
Sample description Target compounds Chromatographic system Derivatization/visualization/detection Reference

Stationary Developing solvent

phase

Bee pollen (of various Polyphenols HPTLC silica A: ethyl-acetate/dichloromethane/ NP reagent and PEG 400; [69]
botanical origin) gel 60 F254 glacial acetic acid/formic acid/water Target compounds visualized as orange,
from Turkey (100/25/10/10/11, v/v/v/v/v). yellow, green, and blue bands, illumination
B: n-hexane/ethyl acetate/glacial acetic under UV light (366 nm)
acid (5/3/1, v/v/v).
Bee pollen of different Polyphenols HPTLC silica A: toluene/ethyl acetate/formic acid (4/ 0.5% NP in ethyl acetate, and 5% PEG 4000 [70]
botanical origin gel 60 F254 7/1, v/v/v) in dichloromethane.
collected from B: ethyl acetate/water/formic acid (17/ Target compounds visualized as orange,
several locations in 2/2, v/v/v) yellow, green, and blue bands under UV light
Serbia (366 nm)
Bee pollen from Spain, Mono- and disaccharides HPTLC silica 148 mg of lactic acid in 20 mL of water, 0.2% napthoresorcinol, 0.4% [71]
Israel, China, and gel 60 F254 40 mL of 2-propanol and 40 mL of diphenylamine, and 4% H2SO4 in 95% of
Romania acetone ethanol.
Target compounds visualized after heating at
100 ◦ C for 2 min
Pollen particles Carotenoids (zeaxanthin, TLC silica gel Gradient development in 15 steps. Mobile Initial inspection under UV light (425 nm) [72]
collected directly cryptoxanthin, β-carotene, 60 phase starting composition: THF/ Recording Raman spectra (250 cm− 1 – 3750
from trees and lutein) methylene chloride/n-hexane (10/90/0, cm− 1) of individual bands after initial
v/v/v). At the end of gradient: n-hexane excitation at 488 nm.
100%.
Pollen particles Neutral lipids (DAG, FFA, TLC silica gel n-hexane/diethyl ether/acetic acid Iodine staining [73]
obtained from plant TAG, SE) 60 (70:30:1, v/v/v)
(Ricinus communis)
Pollen particles Polar lipids (CL, PC, PE, PI, TLC silica gel chloroform/methanol/water Zinzadaze (for phosphorous containing [74]
obtained from plant PG, phosphatidic acid, 60 G254 (65:25:4, v/v/v) - for separation of polar lipids), α-naphthol (for galactolipids),
(Brassica napus L.) phosphatidylserine, lipids Dragendorff’s reagent (for choline
and SPH) containing phospholipids), ninhydrin (for
benzene/diethyl ether/ethyl acetate/ amino containing phospholipids), anthrone
Neutral lipids (DAG, FFA, acetic acid reagent (for sulpholipids).
TAG, SE) (80:10:10:0.2) - for separation of neutral acidic FeCl3 (for neutral lipids)
lipids
Pollen particles Cannabinoids (THC, CBD, Silica gel and Hexane-dioxane (4:1, v/v) – for analysis Fast blue salt B (for cannabinoids), Target [75]
obtained from plant THCA, CBDA). cellulose of cannabinoids. compounds visualized as orange, orange-
(Cannabis sativa L.) Alkaloid type compounds acetone, chloroform, and 25% ammonia yellow- or purple-colored spots.
Flavonoids (160:10:10, v/v/v), or Dragendorff’s reagent, Trabert reagent and
butanol, acetic acid and water (4:1:5, v/ potassium iodoplatinate (for alkaloid type
v/v), or compounds).
isobutanol, fuming HCl and water (7:1:2, AlCl3 solution (for flavonoids)
v/v/v) – for analysis of flavonoids and
alkaloids

CL-cardiolipin, PC-phosphatidylcholine, PE-phosphatidylethanolamine, PI-phosphatidylinositol, PG-phosphatidylglycerol, SPH-sphingomyelin, DAG-diacylglycerols,

FFA-free fatty acids, TAG-triacylglycerol, SE-sterol esters, THC-tetrahydrocannabinol, CBD-cannabidiol, THCA-tetrahydrocannabinolic acid, CBDA-cannabidiolic acid,
NP-natural product, PEG - polyethylene glycol.

Most recently Alimoglu et al. [70] used a simple HPTLC approach in different polarity: I - toluene / ethyl acetate / formic acid (4.7:1, v/v/v)
order to map the main floral constituents of eleven pollen samples from and II - ethyl acetate / water / formic acid (17:2.2, v/v/v). In the first
Turkey. The authors first prepared hydro-ethanolic extracts using 70% case the authors successfully separated less polar phenolics (flavonols,
ethanol - water mixtures, and ultrasonic assisted extraction at 40 ◦ C for flavanons, and isoflavonoids), showing specific patterns with orange
15 min. The ethanol was evaporated and the aqueous residue was sub­ and green colored bands from the rest of the strongly polar compounds.
sequently extracted by ethyl acetate, dichloromethane and n-hexane. In the second case, more polar mobile phase was able to provide uniform
Two developing solvents of different polarity: A - ethyl acetate, separation between strongly polar compounds such as chlorogenic,
dichloromethane, glacial acetic acid, formic acid, and water gallic and ellagic acids, and less polar ones, such as different flavonoid
(100:25:10:10:11, v/v/v/v/v), and B - n-hexane, ethyl acetate, glacial aglycones (apigenin, quercetin, kaempferol) and their glycosides. Prin­
acetic acid (5:3:1, v/v/v), used separately on silica gel plates, increased cipal component analysis performed on chromatographic signals ob­
selectivity of a method and enabled the use of 21 standard compounds tained after capturing images of chromatograms and extensive
for identification of key floral markers. In the most polar, ethyl acetate digitalization, was able to result in the five component model with ob­
sub-extract, the authors found rutin, chlorogenic acid, isoquercitrin, ject scores separating samples that originated mostly from Brassicaceae,
quercitrin, and caffeic acid. In a less polar, dichloromethane fraction, Fabaceae, and Apiaceae families. Small number of samples contained
after separation by solvent system A, authors found predominantly Sophora pollen, Rosaceae, Ranunculaceae, and Chenopodiaceae. All of
present luteolin. Using the solvent system B, they additionally identified them being successfully separated from the rest.
galangin, CAPE, and chrysin (Fig. 3). Quian et al. [72] used HPTLC to screen for the presence of sugars in
Mosić et al. [71] used HPTLC fingerprinting combined with digital bee pollen samples. Hydro-methanolic extracts of five pollen samples
image processing and multivariate data analysis in order to reveal re­ collected from Spain, Israel, China and Romania, were analysed on silica
lationships between 24 bee pollen samples of various botanical origin. In gel, with mobile phase containing 0.15% of lactic acid in a mixture of
that sense the methanolic extracts of pollen samples were first purified water / 2-propanol / acetone (1:2:2, v/v/v). After derivatization with
using solid-phase extraction in order to obtain phenolic fractions. After ethanolic solution containing 0.2% of naphthoresorcinol, 0.4% of
that HPTLC analysis was performed using developing solvents of diphenylamine, and 4% of H2SO4 and heating, the presence of

7
D.M. Milojković-Opsenica et al. Journal of Chromatography B 1188 (2022) 123068

Fig. 3. HPTLC chromatogram of the bee pollen ethyl acetate sub-extracts at 366 nm after derivatization. (A) Developing solvent system: ethyl acetate-
dichloromethane-glacial acetic acid-formic acid-water (100:25:10:10:11, v/v/v/v/v). (B) Developing solvent system: n-hexane-ethyl acetate-glacial acetic acid (5:3:1,
v/v/v). Figure was taken from the G. Alimoglu et al. [70], Copyright licence no: 5180790042982.

monosaccharides (xylose, galactose, 2-D-glucose, frucose, manose, sterol esters, they confirmed the presence of fatty substance X, both in
rhamnose, fructose) and disaccharides was clearly confirmed, contrary pollen and male flowers (Fig. 4). The substance had similar retention as
to propolis samples which lacked sugar components. tre-oleins.
In addition to the application of TLC to analysis of bee pollen, there Similarly, Evans and coworkers [75] used TLC to study the lipid
are few reports on using TLC in analysis of pollen grains directly profile of the inner part and the outer coat of pollen particles of two lines
collected from plants in order to trace botanical origin or elucidate plant of Brassica napus L. For identification of neutral lipids the authors used
metabolic pathways. plain silica gel as a stationary phase and mixture of benzene / diethyl
Schulte et al. [73] used HPTLC in combination with Raman spec­ ether / ethyl acetate / acetic acid (80:10:10:0.2, v/v/v/v) as a devel­
troscopy to study the relationship between content of different carot­ oping solvent. After derivatization with acidic solution of ferric chloride
enoids and botanical origin of pollen grains. The authors used pollen the authors confirmed that the main constituents of both parts (inner
samples from horse chestnut, large-leaved linden, European ash, sallow, and the coat) are triacylglycerols, small amounts of unesterified
mahaleb cherry, and tree of heaven. HPTLC of carotenoids was achieved cholesterol and monoacylglycerol, while the presence of diacylglycerols
using extensive gradient automated development, by lowering polarity or free fatty acids was not confirmed.
of the mobile phase. Raman spectra were recorded after irradiation at Paris, Boucher and Cosson [76] used TLC to identify the main can­
488 nm, at different RF values (0.09, 0.60, 0.81, and 0.91). The average nabinoids, alkaloids and flavonoids in pollen of Cannabis sativa L. grown
of recorded carotenoid spectra was in agreement with spectra obtained under artificial conditions. The maximum content of cannabinoids,
directly (in situ) from the pollen grains, confirming that differences in especially THC and THCA was obtained in plants grown at 24 ◦ C illu­
Raman spectra of grains originate mostly from differences in carotenoid minated for 16 h daily.
content. Additionally, the authors confirmed complex, yet unique re­
lationships between different carotenoid content and botanical origin of 6. Conclusion
studied species. Basically HPTLC, as a separation step, was used as a
helpful tool for additional structure elucidation in the case of in situ Due to the complex nature of bee-products, as well as numerous
Raman spectra with extremely broad peaks originating from very com­ approaches to adulteration such as post-harvesting dilution, inconsistent
plex pollen mixtures. labeling of botanical/geographical origin or bioactivity level, authenti­
Lipids in pollen grains can provide substantial information regarding cation of bee-products is a difficult task and a challenge for any analyst.
the metabolic pathways in plants in different stages of growth and Majority of proposed authentication procedures rely on sophisticated
development. Brown and coworkers [74] separated neutral lipids, techniques that mainly involve combination of several analytical
extracted from both, Castor developing seed endosperm and Castor methods. During last decade, HPTLC has found its place as an adequate
pollen, on TLC. Beside polar lipids, diacylglycerol, free fatty acids and and appropriate technique for quality and authenticity control of

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D.M. Milojković-Opsenica et al. Journal of Chromatography B 1188 (2022) 123068

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Declaration of Competing Interest activity of propolis, J. King Saud Univ. Sci. 32 (1) (2020) 784–790, https://doi.
org/10.1016/j.jksus.2019.02.006.
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The authors declare that they have no known competing financial potential of near infrared spectroscopy for determining the phenolic, antioxidant,
interests or personal relationships that could have appeared to influence color and bactericide characteristics of raw propolis, Microchem. J. 134 (2017)
the work reported in this paper. 211–217, https://doi.org/10.1016/j.microc.2017.06.006.
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Acknowledgement propolis, Anal. Bioanal. Chem. 403 (2012) 2859–2867, https://doi.org/10.1007/
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[26] P. Ristivojević, J. Trifković, U. Gašić, F. Andrić, N. Nedić, Ž. Tešić, D. Milojković-
This work was supported by the Ministry of Education, Science and Opsenica, Ultrahigh-performance liquid chromatography and mass spectrometry
Technological Development of the Republic of Serbia (Contract number (UHPLC-LTQ/Orbitrap/MS/MS) study of phenolic profile of Serbian poplar type
451-03-9/2021-14/200168). propolis, Phytochem. Anal. 26 (2) (2015) 127–136, https://doi.org/10.1002/pca.
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