Accepted Manuscript

Title: Development of a high performance thin layer

chromatography method for the rapid qualification and
quantification of phenolic compounds and abscisic acid in
honeys

Authors: Natalia Stanek, Paweł Kafarski, Izabela

Jasicka-Misiak

PII: S0021-9673(19)30437-6
DOI: https://doi.org/10.1016/j.chroma.2019.04.052
Reference: CHROMA 360198

To appear in: Journal of Chromatography A

Received date: 19 February 2019

Revised date: 17 April 2019
Accepted date: 18 April 2019

Please cite this article as: Stanek N, Kafarski P, Jasicka-Misiak I, Development of

a high performance thin layer chromatography method for the rapid qualification
and quantification of phenolic compounds and abscisic acid in honeys, Journal of
Chromatography A (2019), https://doi.org/10.1016/j.chroma.2019.04.052

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Development of a high performance thin layer chromatography method for

the rapid qualification and quantification of phenolic compounds and

abscisic acid in honeys

Natalia Stanek1, Paweł Kafarski2, Izabela Jasicka-Misiak1

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1
Faculty of Chemistry, Opole University, Oleska 48, 45-052 Opole, Poland;

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2
Faculty of Chemistry, Wroclaw University of Science and Technology, ul. Wybrzeże Wyspiańskiego 27, 50-

370 Wrocław;

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nstanek@uni.opole.pl, izajm@uni.opole.pl, pawel.kafarski@pwr.edu.pl

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* Correspondence: izajm@uni.opole.pl; Tel.: +48-077-452-7115

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Highlights
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 Optimization of chromatographic conditions

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Fingerprint analysis with HPTLC

 Chemical profiles for acacia and lime honeys
 Compositions of polyphenols in honeys
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Abstract
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Honey is a natural product with a complex chemical composition consisting of sugars and other bioactive
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compounds. It is important in many traditional systems of medicine, exhibiting interesting bioactivities, in

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particular antimicrobial, anti-inflammatory and antioxidant effects. Authentication of botanical origin of honeys

is particularly important in this context. Therefore, methods for quality control of honey and detection of its

adulteration are very important.

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A HPTLC method for the quantitative determination of phenolic compounds in honey was developed for the first

time. Seven phenolic compounds were detected and determined quantitatively in lime and acacia honey samples.

The obtained results show that the HPTLC profiles of phenolic compounds and abscisic acid can be useful for

determining the floral origin of honeys. Chromatographic tests were supplemented by statistical analysis (PCA

and HCA) led to the successful separation of acacia and lime honey samples.

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Keywords: Optimization of chromatographic conditions, phenolic compounds, authentication of monofloral

honeys, high-performance thin-layer chromatography, chromatographic fingerprints,

1. Introduction

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Honey is a sweet natural product well known for its nutritional, medicinal and cosmetic properties.

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Chemically it is a very diverse product , and its composition depends strongly on the type and species of the

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plant from which the bees collect nectar. The honey industry produces a wide variety of honey from bright to

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dark; in liquid, creamed or solid form; and of the monofloral or multifloral variety. The high nutritional value of

monofloral honey, resulting from a specific, consistent chemical composition, increases its cost compared to that

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of multifloral honey and thus leads to more frequent adulteration of these food products [1].

Two of the most popular and widely consumed in Europe monofloral honeys are acacia (Robinia
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pseudoacacia) and lime (Tilia spp.) honey. These honeys are fairly light in colour, almost transparent, with a
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floral aroma. Acacia is known for its mild and delicate floral flavour, while lime honey has an intense, sweet
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flavour with a touch of bitterness. The characteristics of these honeys match general consumer preferences,

which means that consumers are more likely to buy these products and are willing to pay a high price for them.
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However, purchasers expect a high-quality product with good properties. Most importantly, monofloral honey

should be designated according to the floral or plant source and should be derived wholly or mainly from that
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source. However, industry standards are not recognized by all producers of honey. Some producers blend honeys

from different origins to obtain a honey that meets all the strict requirements of their buyers or add the required
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amount of pollen. Traditionally, classification of the floral origin of honey is determined by

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melissopalynological analysis. This method is based on the identification of pollen by microscopic examination.

Honey is considered monofloral when the content of pollen from a single species exceeds 45% of the total pollen
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content [2]. However, there are some problems related to this method. One limitation of the method is that

honeys, for example, from Robinia and Tilia contain pollen at low levels (less than 20,000 pollen grains per 10

grams of honey) [3,4]. Therefore, classifying these honeys as unifloral based on a melissopalynological analysis

can be very difficult and sometimes impossible. Alternative analytical methods for honey authentication are

based on the identification of chemical marker compounds , such as phenolic compounds [5,6]. Individual

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varieties of honey have different compositions of polyphenols in different proportions, and some of these

compounds can be considered floral markers of the honey origin. However, proposing a honey marker is not

helpful for the determination of the botanical origin of honey samples with a similar phenolic compound

composition. For example, abscisic acid was detected in lime and acacia honeys at similar concentrations [7,8].

In this case, the sources of information are not the presence of individual chemical compounds, but the mutual

quantitative relations of the components of the fraction separated using a specific procedure. Therefore, we can

expect more reliable authentication results by creating chemical profiles for these honeys. A chemical fingerprint

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is a pattern characteristic to a given honey type. Among the numerous analytical methods, fingerprint analysis

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with TLC is of particular interest [9-11].

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Thin layer chromatography is a classic technique used in natural product analysis, especially for the

profiling of plant extracts [12-15]. This method is simple, rapid and inexpensive and ensures low consumption of

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solvents and reagents. TLC has several important advantages over other chromatographic techniques, such as the

ability to analyse many samples at the same time and an analysis that does not require complicated

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instrumentation. However, its low sensitivity and poor resolution and reproducibility resulted in the decreased
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use of this technique in recent times. High performance thin layer chromatography (HPTLC) as a greatly
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advanced form of traditional TLC has increased significantly in popularity over the past decades and has now
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developed into a very popular analytical technique, in particular for natural product research HPTLC uses a

higher quality plate than that used in TLC and instrumentation with automated sample application and
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densitometric scanning. The high quality and continual improvement of HPTLC instrumentation have improved

the sensitivity, precision and accuracy of the results. This technique is based on a chromatographic fingerprint,
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i.e., the chromatographic pattern of the chemically characteristic components present in an extract.

Chromatographic fingerprints can successfully demonstrate both “similarities” and “differences” between
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various samples of honey [16,17]. Therefore, HPTLC can be considered an efficient tool for assessing the origins
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of honeys. Furthermore, HPTLC is suitable for not only qualitative abut also quantitative analysis when

combined with densitometry. The components are separated on the plate, and then, the optical density of the
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separated spots is measured. The sample concentrations are determined by comparison with standard curves

generated from standards chromatographed under the same conditions. To obtain reliable analysis results, the

method should be validated using routine parameters such as linearity, precision, limit of detection and limit of

quantitation. Then, published results can be considered comparable with other data gathered using the same

standardized analytical procedure. To verify the origin, authenticity and quality of acacia and lime honey, a

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simple and rapid HPTLC method was applied to determine the honey composition. In addition, principal

component analysis (PCA), as an unsupervised chemometric technique, was applied to extract the full

information from chromatograms, confirm the similarity/dissimilarity among samples, and identify characteristic

markers of the botanical origin of the honey.

In this study, the focus was on developing a new and simple analytical high-performance thin-layer

chromatographic method for distinguishing and authenticating of Polish acacia and lime honeys on the basis of

their phenolic compound content.

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2. Materials and Methods

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2.1. Chemicals and materials

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All chemicals and reagents used were of analytical grade. Methanol, chloroform, ethyl acetate, formic acid and

aluminium chloride were purchased from POCH S.A. (Gliwice, Poland). The standards chrysin, myricetin,

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quercetin, naringenin, caffeic acid, cinnamic acid, chlorogenic acid, ferulic acid and (±)-abscisic acid were

purchased from Sigma-Aldrich (Poznań, Poland). Amberlite XAD-2, used as an adsorption resin in the

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extraction step, was obtained from Supelco (Bellefonte, PA, USA). HPTLC analyses were performed on 20 cm x
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10 cm HPTLC silica gel 60 plates from Merck (Darmstadt, Germany).
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2.2. Honey samples
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Thirty honey samples, 13 acacia (Robinia pseudoacacia L.) and 17 lime (Tilia L.), were analysed. Their

botanical origins were confirmed by pollen analysis. The characteristics of the honey samples are presented in
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Table 1.

2.3. Extraction of phenolic compounds

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Phenolic compounds extraction was performed as described previously [16] with some modifications. The honey
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samples (10 g) were thoroughly mixed with 50 mL of acidified water (HCl, pH=2.0). The honey solution was
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filtered and transferred to a glass column (30 x 2 cm). Amberlite XAD-2 (25 g) (Supelco, Bellefonte, PA, USA,

pore size: 9 nm, particle size: 0.3–1.2 mm), used as an adsorption resin. After 30-45 min, the column was
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washed with 100 mL of acidified water (HCl, pH=2.0) and subsequently rinsed with 125 mL of distilled water to

remove all sugars and other polar components. The phenolic compounds were eluted with 200 mL of methanol.

The methanolic extract was evaporated under reduced pressure at 40°C, and the residue was then dissolved in 2

mL of methanol (HPLC-grade) and analysed by HPTLC.

2.4. High-performance thin layer chromatography

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The honey extract (20 µL) and standard solutions (10 µL or 5 µL) were applied to HPTLC plates using an

automatic HPTLC application device (Linomat 5, CAMAG, Muttenz, Switzerland). Chromatographic separation

was performed on 20 cm x 10 cm (0.25-thick) HPTLC plates (HPTLC silica gel 60, Merck). A mixture of

chloroform-ethyl acetate-formic acid (5:4:1, (v/v/v)) was used as the mobile phase. Visualization was performed

under UV light at 254 nm and at 366 nm or by spraying with 1% methanolic AlCl 3. The obtained

chromatographic images were analysed using HPTLC software (visionCATS, CAMAG).

2.5. Method of validation

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Quantitative evaluation was performed in the absorbance mode at 366 nm. The proposed method was validated

on the basis of the linearity, precision, detection limit and quantitative limit. A summary of the validation

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parameters is presented in Table 2.

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2.5.1. Linearity

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The linearity was evaluated by analysing standard solutions with concentrations from 5-400 µg/ml. The obtained
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working standard solutions (10 µL for chrysin, myricetin, naringenin, and caffeic acid and 5 µL for quercetin,

chlorogenic acid, and ferulic acid) were applied to the same plate to prepare five-point linear calibration curves.
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The analyses were performed in triplicate. In Fig 1, an example chromatogram and series of densitograms
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validated for quercetin is presented.

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2.5.2. Precision

The repeatability of the sample application and measurement of the peak area were determined by analysing
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three replicates of 100 µg/ml standard solutions under the same conditions on the same day (intraday precision)

and over a period of one week (interday precision). The precision was expressed as the relative standard
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deviation (RSD [%]).

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2.5.3. Limit of detection and limit of quantitation

The limit of detection (LOD) and limit of quantitation (LOQ) were based on the calibration curves. The
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calibration curves were prepared using standard solutions with concentrations in the range of the detection limit.

The LOD was calculated as LOD= 3.3*SD/a

The LOQ was calculated as LOQ= 10*SD/a

where “SD” is the standard deviation and “a” is the slope of the calibration curve.

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 2.6. Multivariate statistical analysis

The statistical analyses were performed using the software Statistica 13.1 (StatSoft). The method used for

multiple comparisons was the least significant difference test, and statistical significance was set at p≤0.05.

Classification technique principal component analysis (PCA)was used to compare the concentrations of

identified compounds in the honey samples.

3. Results and Discussion

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3.1. High-performance thin layer chromatography profile

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The aim of this study was to identify and quantify abscisic acid and the phenolic compounds characteristic of

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acacia and lime honey [18]. For this purpose, HPTLC fingerprint analysis was performed. Thirty honey samples

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were investigated, including 13 acacia and 17 lime honey samples (Table 1). The chromatographic results are

presented as electronic images in Figs 2-5. The identification of phenolic compounds and abscisic acid was

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based on Rf values (position of bands and colour) of standards and honey extracts . The HPTLC chromatograms

indicate similarities and differences in the composition of the identified compounds between the acacia and lime
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honey samples. The pattern for acacia honey is dominated by yellow and blue bands, with major bands at Rf
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0.03, 0.08, 0.15, 0.20, 0.31, 0.48, 0.50, 0.55, 0.61, 0.65, and 0.76 (Figs. 2-3). The pattern for lime honey is
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characterized by yellow, blue and black bands. The lime honey samples present notable bands at R f 0.03, 0.08,

0.16, 0.18, 0.46, 0.49, 0.53, 0.56, 0.60, 0.67, 0.74, and 0.82 (Figs. 4-5). Chlorogenic, caffeic, and ferulic acid and
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myricetin were identified in both floral varieties of honey. Additionally, quercetin and cinnamic acid were found

in both types of honey but not in all samples. In lime honey, the most prominent band is blue and located at Rf
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0.60, corresponding to ferulic acid (134.5-1872.7 µg/100 g of honey). Additionally, only lime honey samples are

characterized by the presence of black spots, such as that corresponding to abscisic acid (Rf = 0.56); dull brown
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spots, such as that corresponding to chrysin (Rf = 0.82); and yellow spots, such as that corresponding to
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naringenin (Rf = 0.67).

The concentrations of the selected standard compounds in the honey samples are given in Table 3. Excitation
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was commonly performed using radiation at 366 nm. This method of detection is the most sensitive of all

HPTLC detection methods and has been used for quantification [19]. Some phenolics can be detected by

quenching the adsorbent fluorescence. After ultraviolet irradiation, the emission of the adsorbent is reduced in

regions where UV-active compounds absorb UV light. These regions appear as dark spots [19, 20]. In this study,

abscisic acid and cinnamic acid quenched the fluorescence and appeared as dark spots, which enabled their

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detection, but their quantification was difficult. However, many phenolic compounds possess the ability to

fluoresce and appear as brightly coloured zones. Additionally, these compounds can be visualized by spraying

the plate with a specific reagent. Under UV light, flavones such as chrysin appear dull brown but fluoresce

yellow after being sprayed with 1% methanolic AlCl3. The obtained phenolic profiles allow the clear

differentiation between acacia and lime honey. The main difference between the phenolic profiles of lime and

acacia honey is the following: in the former, ferulic acid is a notable band compared to the bands of the other

phenolic compounds, while in the latter, this compound creates a band of less intensity. Furthermore, the lime

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honey samples were characterized by additional bands corresponding to abscisic acid, chrysin and naringenin,

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whereas in the case of acacia honey, these bands were not observed. Careful evaluation of the patterns involving

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phenolic compounds and abscisic acid could indicate the botanical origin of honey.

3.2. Multivariate statistical analysis

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To illustrate the relationship between the concentrations of the identified compounds and the origin of honey,

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principal component analysis (PCA) was applied. PCA was conducted to evaluate the effect of the identified
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phenolic compounds on the honey origin. As seen Fig 6A, each honey sample is characterized by two values,

one along PC1 and another along the PC2. Two principal components explained 54.21% of the total variance
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(PC1 33.97% and PC2 20.24%). Fig 6A presents two different groups, each corresponding to a botanical group:
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acacia or lime honey. The acacia and lime honey samples partially overlap, but their separation can be observed.

The greatest influence on such a grouping involved the concentrations of caffeic and ferulic acids, chrysin (lime
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honey) and quercetin (acacia honey). Regarding the correlation loadings presented in Fig 6B, the quercetin had a

positive correlation with PC1 and caffeic, ferulic acids and chrysin showed a negative correlation with PC1. This
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means, for instance, that the variables quercetin, chrysin, caffeic and ferulic acids separate the acacia honey

samples from lime honey samles. The acacia honey are characterized by having high values of quercetin and low
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concentration of chrysin, ferulic and caffeic acid. Moreover, the model interpration suggest that lime honey
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samples have high value of chrysin, ferulic and caffeic acids and low value quercetin. The results of PCA

produced a good classification of the honey samples according to their botanical origin. To conclude, high
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concentration of caffeic, ferulic and chrysin were characteristic for lime honeys and positioned on the negative

PC1-score while high value of quercetin was characteristic for acacia honeys and positioned on the positive PC1-

score.

4. Conclusion

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An HPTLC method for the quantitative determination of phenolic compounds in honey was developed for the

first time. Seven phenolic compounds were detected and determined quantitatively in honey samples (except

cinnamic acid and abscisic acid). Furthermore, in our study, the HPTLC fingerprints of 30 honey samples were

visually compared with one other. The present results show that the HPTLC profiles of phenolic compounds and

abscisic acid can be useful for determining the floral origin of honeys. The proposed method is rapid, simple and

precise. Additionally, the combination of HPTLC fingerprints with statistical data analysis (PCA ) led to the

successful separation of acacia and lime honey samples. This approach shows the great potential of this method

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for assessing the botanical origin of food products.

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Acknowledgements

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This work was supported by The National Science Centre of Poland in the frame of Grant NO:

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2014/15/B/N29/02182.

Conflict of Interest

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The authors declare that they have no conflict of interest.
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Tables

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Table 1. Floral origin of analysed honey samples.

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Floral origin (% of pollen)

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Acacia honey (Robinia pseudoacacia L.) Lime honey (Tilia L.)

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Sensory Sensory
% of pollen Colour Intensity characteristics % of pollen Colour Intensity characteristics
pH pH
* *
>30% [mAU]
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consistency) consistency)

A1 30.5% 0.008 White, solid 4.0 L1 27.9% 0.102 Yellow, liquid 4.3
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A2 32.5% 0.021 Pale yellow, liquid 4.2 L2 22.8% 0.107 Yellow, liquid 4.8
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A3 31.8% 0.018 Pale yellow, liquid 4.2 L3 24.7% 0.159 Yellow, liquid 4.1
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A4 37.6% 0.017 Pale yellow, liquid 4.0 L4 23.8% 0.103 Yellow, liquid 4.4

A5 30.4% 0.037 Yellow, liquid 4.1 L5 25.1% 0.083 Yellow, liquid 4.5
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A6 31.6% 0.026 Pale yellow, liquid 4.0 L6 25.9% 0.076 Pale yellow, solid 4.6

A7 31.8% 0.019 White, solid 4.0 L7 26.3% 0.083 Yellow, liquid 4.4

A8 34.9% 0.080 Pale yellow, solid 3.9 L8 49.0% 0.079 Yellow, liquid 4.3

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A9 31.7% 0.010 Pale yellow, solid 3.8 L9 20.0% 0.077 Yellow, liquid 4,1

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A10 37.6% 0.015 Pale yellow, liquid 3.8 L10 20.1% 0.141 Yellow, solid 4.0

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A11 30.7% 0.025 Pale yellow, liquid 4.0 L11 25.6% 0.102 Yellow, liquid 4.2

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A12 30.8% 0.010 Pale yellow, liquid 4.0 L12 45.0% 0.165 Pale yellow, liquid 4.1

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A13 35.7% 0.037 Yellow, liquid 4.0 L13 30.3% 0.067 Yellow, liquid 4.1
ED L14 20.7% 0.110 Pale yellow, solid 4.0

L15 24.3% 0.140 Yellow, liquid 4.7

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L16 23.3% 0.100 Yellow, liquid 4.3

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L17 21.2% 0.077 Yellow, liquid 4.2

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*
value determined using the Polish Quality Standard PN-88/A-77
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Table 2. Validation parameters of phenolic compounds.

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Linearity range LOD LOQ RSD [%] Scanning
Standards Regression equation R2
[µg/ml] [µg/ml] [µg/ml] Inter-day Intra-day [nm]

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Caffeic acid 12.5-200 y = 0.1033x +0.6625 0.9997 3.7 11.1 0.92 2.80 366
Chlorogenic acid 5-200 y = 0.0344x +0.3060 0.9967 6.7 20.3 1.13 3.66 366

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Ferulic acid 5-200 y = 0.0482x +0.7369 0.9966 6.8 20.7 0.98 3.47 366

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Chrysin 25-400 y = 0.0149x +0.5417 0.9991 11.1 33.6 2.55 4.17 366
Myricetin 12.5-200 y = 0.1953x +0.6042 0.9971 4.2 12.8 1.13 4.31 366
Naringenin ED12.5-200 y = 0.0682x +0.9306 0.9985 8.2 24.9 1.79 5.11 366
Quercetin 12.5-200 y = 0.1046x +0.0278 0.9991 11.4 34.4 1.67 4.81 366
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Table 3. Quantitative determination of phenolic compounds in acacia and lime honey samples.

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Honey Phenolic acids Flavonoids
(µg/100g honey ±SD, n=3) ( µg/100g honey ±SD, n=3)

N
Acacia CA ChA FA Ch M N Q
A1 nd 46.7 ±9.6 nd nd nd nd 123.5 ±10.3

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A2 nd 153.8 ±9.0 nd nd 24.9 ±4.9 nd 210.5 ±10.9

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A3 nd 152.3 ±7.6 17.2 ±2.2 nd 14.4 ±1.1 nd 196.2 ±8.8
A4 nd 51.6 ±5.4 85.7 ±2.9 nd 29.8 ±5.4 nd nd
A5 nd ED 37.0 ±9.3 35.9 ±4.0 nd 79.0 ±3.9 nd 185.7 ±11.9
A6 nd 56.4 ±5.7 nd nd 15.3 ±5.9 nd nd
A7 nd 17.2 ±1.5 82.6 ±4.4 nd nd nd nd
PT
A8 nd 120.3 ±9.2 78.4 ±1.5 nd 37.4 ±8.5 nd 182.8 ±12.3
A9 82.4 ±5.6 85.5 ±8.5 124.1 ±9.7 nd 25.7 ±6.3 nd 145.5 ±10.8
A10 nd 36.0 ±3.1 56.7 ±5.9 nd 36.7 ±5.6 nd nd
E

A11 nd 18.6 ±1.3 110.6 ±7.5 nd 75.0 ±11.6 nd nd

CC

A12 nd nd nd nd nd nd 240.2 ±10.2

A13 nd 123.3 ±8.2 47.3 ±4.0 nd nd nd nd
Lime
A

L1 nd 23.0 ±4.4 1746.8 ±147.1 260.6 ±25.5 32.0 ±1.5 nd nd

L2 13.3 ±2.6 27.3 ±4.6 782.8 ±70.14 676.7 ±52.3 25.6 ±0.4 nd nd
L3 21.1 ±3.4 79.7 ±6.2 1298.0 ±96.1 525.7 ±36.7 307.8 ±22.8 nd nd
L4 nd 51.6 ±6.0 807.7 ±46.2 904.9 ±37.7 254.0 ±16.3 nd nd
L5 49.1 ±3.6 219.2 ±31.0 1037.3 ±116.4 723.7 ±62.0 112.7 ±14.1 150.9 ±30.2 nd
L6 nd 33.1 ±2.5 639.0 ±88.1 nd 46.2 ±7.1 nd nd

14
 R I
SC
L7 40.4 ±3.2 72.9 ±8.9 919.7 ±99.8 710.2 ±75.4 91.7 ±9.9 nd nd
L8 34.1 ±3.6 140.7 ±15.0 741.3 ±20.9 nd 43.1 ±9.2 nd nd

U
L9 43.6 ±2.7 nd 1184.6 ±53.8 nd 283.7 ±13.4 nd nd
L10 27.8 ±4.5 15.7 ±1.6 1512.4 ±146.1 368.0 ±54.2 96.6 ±13.6 nd nd

N
L11 nd 18.6 ±1.8 1872.7 ±116.6 nd 315.5 ±13.4 nd nd

A
L12 nd 418.8 ±64.5 304.2 ±28.2 76.0 ±10.7 753.5 ±66.2 nd nd
L13 nd 12.8 ±4.5 571.2 ±34.7 206.9 ±30.4 40.6 ±7.7 nd nd

M
L14 33.2 ±3.4 210.5 ±42.8 867.2 ±166.2 817.7 ±80.3 54.9 ±3.3 nd nd
L15 30.4 ±6.9 82.6 ±16.4 883.1 ±81.7 126.4 ±25.4 125.2 ±8.0 nd nd
L16
L17
nd
ED
45.6 ±8.1
nd
69.5 ±2.6
134.5 ±25.2
285.6 ±63.6
nd
542.4 ±35.5
14.1 ±2.4
14.7 ±3.0
nd
nd
nd
nd
ChA- chlorogenic acid, M- myricetin, CA- caffeic acid, Q- quercetin, FA- ferulic acid, N- naringenin, Ch- chrysin, nd- not detected.
E PT
CC
A

15
Figures

T
R IP
SC
Fig. 1. HPTLC chromatogram and series of densitograms of quercetin.

U
N
A
M
ED

Fig. 2. HPTLC chromatogram of phenolic compounds at 254 nm in acacia honeys (A1-A13). Phenolic
PT

compounds are identified as: ChA- chlorogenic acid, M- myricetin, CA- caffeic acid, Q- quercetin, FA- ferulic

acid, CiA- cinnamic acid.

E
CC
A

16
 T
IP
Fig. 3. HPTLC chromatogram of phenolic compounds at 366 nm in acacia honeys (A1-A13). Phenolic

compounds are identified as: ChA- chlorogenic acid, M- myricetin, CA- caffeic acid, Q- quercetin, FA- ferulic

R
acid, CiA- cinnamic acid.

SC
U
N
A
M

Fig. 4. HPTLC chromatogram of phenolic compounds and abscisic acid at 254 nm in lime honeys (L1-L17). The
ED

compounds are identified as: ChA- chlorogenic acid, M- myricetin, CA- caffeic acid, AA- abscisic acid, Q-

quercetin, FA- ferulic acid, N- naringenin, CiA- cinnamic acid, Ch- chrysin.
E PT
CC
A

Fig. 5. HPTLC chromatogram of phenolic compounds and abscisic acid at 366 nm in lime honeys (L1-L17). The

compounds are identified as: ChA- chlorogenic acid, M- myricetin, CA- caffeic acid, AA- abscisic acid, Q-

quercetin, FA- ferulic acid, N- naringenin, CiA- cinnamic acid, Ch- chrysin.

17
 T
IP
Fig. 6. Principal component analysis of honey samples based on phenolic compounds concentrations. - A.

R
Scores: A1-A13- acacia honey samples, L1-L17- lime honey samples, B. Loadings: ChA- chlorogenic acid, M-

SC
myricetin, CA- caffeic acid, Q- quercetin, FA- ferulic acid, N- naringenin, Ch- chrysin

U
N
A
M
ED
E PT
CC
A

18