Simultaneous determination of carvacrol and thymol in bee pollen by using a simple and efficient solvent extraction method and gas chromatography-mass spectrometry

doi:10.1016/j.jpba.2020.113124

Journal of Pharmaceutical and Biomedical Analysis

Volume 181, 20 March 2020, 113124

https://doi.org/10.1016/j.jpba.2020.113124 Get rights and content

Highlights

•
Thymol and carvacrol were simultaneously analysed in bee pollen by using GC–MS.
•
The proposed sample treatment has shown to be effective (mean recoveries ≥ 90%).
•
Separation was achieved in under 21 min by means of a ZB-Wax column.
•
Matrix did not affect to the MS signals of both compounds.
•
Residues of thymol and carvacrol (LOQ-57 μg/kg) were found in some samples.

Abstract

A novel method is proposed to determine residues of carvacrol and thymol in bee pollen by means of gas chromatography coupled to mass spectrometry. This is an efficient and simple sample treatment (with average analyte recoveries between 90% and 104%) involving solvent extraction with hexane followed by evaporation. There is no need for any additional clean-up step, as the matrix did not affect determination of mass spectrometry for either compound. The chromatographic conditions are also optimized: a ZB-WAX column is employed, helium is the carrier gas at a flow rate of 1.1 mL/min, and a temperature program is included, allowing baseline separation of both compounds in less than 21 min. The method is fully validated in terms of selectivity, limits of detection and quantification, matrix effect, linearity, precision and trueness. Results show that not only is it selective, but that it also displays a wide linearity range (limit of quantification-1000 μg/kg), good precision (relative standard deviation values lower than 8%) and sensitivity (limits of detection and quantification lower than 15 μg/kg). Finally, several bee pollen samples are analysed, and thymol and carvacrol residues are found at low concentrations (limit of quantification-57 μg/kg) in some cases.

Graphical abstract

Introduction

Essential oils are among the natural products that have been used to fight against the Varroa destructor mite, which is considered to be one of the most destructive pests for the honey bee. They represent the preferred alternative by the food industry and consumers rather than synthetic acaricides [1]. In particular, thyme (Thymus vulgaris) oil, which is rich in thymol and carvacrol, widely used in industry as antimicrobial agents [2,3], has been employed by several beekeepers [[4], [5], [6], [7]]. In fact, thymol is currently considered a pesticide by the European Union [8], and both compounds present a broad spectrum of therapeutic activities, such as the anti-bacterial, anti-fungal, anti-parasitic, anti-oxidant, and anti-inflammatory [9,10]. Consequently, it may be surmised that residues of these compounds might accumulate in different bee products (beeswax, honey and bee pollen) after application of essential oil treatment [11]. Moreover, monitoring thymol and carvacrol in bee products could be of interest, not only to verify that thyme oil has been adequately administered, but also to check the potential presence of these bioactive compounds in foods to be consumed by humans. Most studies related to determining thymol and/or carvacrol focused on honey [[4], [5], [6],[12], [13], [14], [15], [16]], with scant consideration being given to other bee products, such as pollen. The latter is attracting particular attention as a functional food/food supplement for human consumption due to its high content of bioactive compounds like essential amino acids, anti-oxidants, vitamins, minerals and lipids [17]. This is related with a wide range of biological activities, including the anti-fungal, anti-microbial, anti-viral, hypolipidemic, anti-atherosclerotic, anti-inflammatory and cerebrovascular [17]. To the best of our knowledge, in only two surveys were thymol residues reported in bee pollen [18,19], yet no studies are available on determining carvacrol in this matrix. In both studies, a sample preparation based on a quick, easy, cheap, effective, rugged and safe method (QuEChERS) was used, but no specific data was provided. Moreover, the extraction procedure was taken from a previous study investigating a large number of pesticides in other produce [20]. Once again, no mention was made of thymol and/or carvacrol being included among the pesticides examined or of the proposed methodology being optimized for bee pollen analysis. Thymol and carvacrol were analysed more frequently in other bee related matrices like honey, beeswax or honeybees [[4], [5], [6], [7],12,14,[13], [14], [15], [16],21]. Several sample treatments were employed in these studies: solid-phase extraction, solid-phase microextraction, dispersive liquid-liquid microextraction, QuEChERS, sorptive extraction and simple extraction in a suitable solvent. These matrices, however, are totally different from bee pollen, and many of the above-mentioned sample treatments could not be easily adapted for this matrix. Therefore, in view of the few publications concerning thymol and carvacrol analysis in bee pollen, and due to our experience in analysing phenolic compounds (resveratrol and piceid isomers) in this matrix [22], we considered appropriate a simple solvent extraction as an alternative to QuEChERS. This is in line with current trends in sample preparation techniques. These focus on simplifying treatment in order to reduce not only costs but also the number/amount of reagents and time, which are among the principles of green analytical chemistry [23]. In addition, according to the related bibliography, the most common technique for determining thymol and carvacrol was gas chromatography (GC) [[4], [5], [6], [7],12,14,15], although liquid chromatography [6,13,16] and electrochemical methods [21] were also employed. Regarding detection systems, some articles reported the use of flame-ionization detectors [4,12,15]; nowadays, however, mass spectrometry (MS) detectors are gaining attention [5,7,14,15]. In our case, analyses were performed in a GC–MS system.

The aim of this study was to propose a specific analytical methodology by means of GC–MS in order to determine simultaneously thymol and carvacrol in bee pollen. We have optimized extraction and determination procedures with the aim of providing good recoveries with the simplest procedure possible, whilst minimizing the potential matrix effect in all cases. To the best of our knowledge, this is the first study in which carvacrol has been determined in this matrix, and in which there has been specific development for the simultaneous determination of thymol and carvacrol in bee pollen. Further aims of the study focused on validating the proposed method in accordance with current European legislation, as well as analysing different bee pollen from two Spanish regions (Castilla la Mancha and Galicia).

Section snippets

Reagents and materials

Thymol (Purity > 98.5%) and carvacrol (Purity 98.0%) standards, and ethanol were purchased from Sigma Aldrich Chemie Gbmh (Steinheim, Germany). Hexane and acetonitrile were obtained from Panreac (Barcelona, Spain). It must be remarked that all solvents used were of analytical grade. A vibromatic mechanical shaker and a drying oven, both supplied by J.P. Selecta S.A. (Barcelona, Spain), a vortex mechanical mixer from Heidolph (Schwabach, Germany), a 5810 R refrigerated bench-top Eppendorf

Optimization of the sample treatment

As mentioned in the Introduction, we decided to use a solvent extraction for sample treatment: The first step of the optimization process was to select the most suitable solvent to extract thymol and carvacrol from the bee pollen. According to the literature regarding thymol and carvacrol determination in other food matrices, especially in plants and essential oils, ethanol, hexane and chloroform were usually selected. [[25], [26], [27], [28], [29]]. We decided, however, to avoid the use of

Conclusions

This is the first time a specific GC–MS based method has been developed, optimized and validated for simultaneously determining thymol and carvacrol in bee pollen. A rapid, simple, and efficient sample treatment based on a solvent extraction with hexane has been proposed. No matrix effect has been found in the case of both analytes, which is an additional advantage of this procedure, as quantification can be performed with reference standards in solvents. Separation has been achieved in under

Funding

This work was supported by the Spanish “Ministerio de Economía y Competitividad” and the “Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (Project number RTA2012-00076-C02-02).

CRediT authorship contribution statement

Ana M. Ares: Conceptualization, Methodology, Investigation, Validation, Writing - original draft. María J. Nozal: Conceptualization, Methodology, Validation, Visualization. José L. Bernal: Conceptualization, Funding acquisition, Project administration, Visualization. José Bernal: Conceptualization, Project administration, Resources, Supervision, Visualization, Writing - review & editing.

Declaration of Competing Interest

None.

Acknowledgements

Authors thank David Rixham (White Rose English School, Valladolid, Spain) for performing the revision of the English language/grammar of the manuscript. In addition, authors thank E. Pérez and J. Tapia for their technical assistance.

References (31)

M. Hudaib et al.
GC/MS evaluation of thyme (Thymus vulgaris L.) oil composition and variations during the vegetative cycle
J. Pharm. Biomed. Anal.
(2002)
I.A. Al-Saleh et al.
Levels of selenium DL-α-tocopherol, DL-γ-tocopherol, thymoquinone and thymol in different brands of Nigella sativa seeds
J. Food Anal.
(2006)
M.J. Nozal et al.
Extraction of thymol, eucalyptol, menthol and camphor residues from honey and beeswax. Determination by gas chromatography with flame ionization detection
J. Chromatogr. A
(2002)
N. Tonello et al.
Square wave voltammetry with multivariate calibration tools for determination of eugenol, carvacrol and thymol in honey
Talanta
(2016)
P. Viñas et al.
Liquid chromatographic determination of phenol, thymol and carvacrol in honey using fluorimetric detection
Talanta
(2006)
K. Kasiotis et al.
Chromatographic determination of monoterpenes and other acaricides in honeybees: prevalence and possible synergies
Sci. Total Environ.
(2018)
F.Q. Pires et al.
Use of mixture design in drug-excipient compatibility determinations: thymol nanoparticles case study
J. Pharm. Biomed. Anal.
(2017)
M. Shakeri et al.
Carvacrol and astaxanthin co-entrapment in beeswax solid lipid nanoparticles as an efficient nano-system with dual antioxidant and antibiofilm activities
LWT- Food Sci. Technol.
(2019)
J.I. Cacho et al.
Evaluation of three headspace sorptive extraction coating for the determination of volatile terpenes in honey using gas chromatography-mass spectrometry
J. Chromatogr. A
(2015)
A.M. Ares et al.
Extraction and determination of bioactive compounds from bee pollen
J. Pharm. Biomed. Anal.
(2018)

A. Gałuszka et al.

The 12 principles of green analytical chemistry and the SIGNIFICANCE mnemonic of green analytical practices

Trends Analyt. Chem.

(2013)

E.M. Napoli et al.

Screening of the essential oil composition of wild Sicilian thyme

Biochem. Syst. Ecol.

(2010)

J. Ivanovic et al.

The analysis of simultaneous clove/oregano and clove/thyme supercritical extraction

J. Supercritic. Fluid.

(2011)

V. Rowshan et al.

Influence of storage conditions on the essential oil composition of Thymus daenensis Celak

Ind. Crops Prod.

(2013)

R. Requena et al.

Study of the potential synergistic antibacterial activity of essential oil components using the thiazolyl blue tetrazolium bromide (MTT) assay

LWT-Food Sci. Technol.

(2019)

Cited by (16)

Second-order electrochemical data generation to quantify carvacrol in oregano essential oils
2022, Food Chemistry
Citation Excerpt :
The most common CAR determination methods are manly based on chromatographic techniques, such as gas chromatography (GC) and liquid chromatography (LC). For GC, different detection systems were reported, such as flame ionization detector (GC-FID) (Daneshfar et al., 2016; Khajeh et al., 2015; Sereshti et al., 2011; Kiyanpour et al., 2009) and mass spectrometry (GC–MS) (Ghiasvand et al., 2011; Cacho et al., 2015; Jiménez-Salcedo & Tena, 2017; Bernal et al., 2020; Ares et al., 2020). On the other hand, LC coupled with triple quadrupole mass spectrometry or fluorescence detection were also described for the CAR determination (Oller-Ruiz et al., 2017; Viñas et al., 2006).
A novel analytical method using voltammetric second-order modeling based on multivariate curve resolution-alternating least-square (MCR-ALS) is presented for the first time for the quantitation of carvacrol (CAR) in oregano essential oils (OEO). The second-order cyclic voltammetry data were generated on the basis that CAR shows a diffusional system. Thus, the scan rate (v) was used as a second instrumental mode and cyclic voltammograms at different v were acquired for a single sample, generating the second-order data. CAR determination was performed in presence of thymol, included as a potential interferent. Results demonstrated that MCR-ALS successfully exploited the second-order advantage and the recoveries were not statistically different than 100%. The limits of detection and quantitation were estimated using the MCR-ALS which were 6.27 × 10⁻⁵°mol°L^−1°and 1.90 × 10^−4°mol L⁻¹, respectively. Finally, the developed methodology was implemented to quantify of CAR in OEO samples.
Presence, persistence and distribution of thymol in honeybees and beehive compartments by high resolution mass spectrometry
2021, Environmental Advances
Citation Excerpt :
The determination of this compound is rising in different honeybee products. Ares et al determined residues of carvacrol and thymol in bee pollen by gas chromatography (GC) coupled to mass spectrometry (GC-MS) with hexane as a solvent extraction followed by evaporation without clean-up step (Ares et al., 2020), Kasiotis et al analyzed thymol in bees by GC coupled to tandem mass spectrometry (GC-MS/MS) with modified QuEChERS technique (Karasali et al., 2014). Nozal et al analyzed thymol residues from honey and beeswax by GC coupled to flame ionization detector (GC-FID) (Nozal et al., 2002).
Thymol has been considered as an organic varroacide with less toxicity than synthetic chemicals such as amitraz, coumaphos or tau-fluvalinate. The present study evaluates the presence, persistence and distribution of thymol during a five-month sampling period; before, during and after the application of thymol treatment in field conditions. Moreover, its impact on the development of bee brood was also studied at different development stages which consist of larvae, prepupae, pupae and adult honeybee. Samples of beebread (processed pollen), honey, beeswax, adult bees, pupae, prepupae and larvae were analysed by gas chromatography coupled to time of flight mass spectrometry for an accurate identification. Adult bees were the samples with the highest concentration level of thymol during treatment application (Thymovar® strips, 30 gr of thymol per hive) by the beekeeper, however they showed 69% of thymol dissipation three months after treatment. Regarding the bee brood, concentrations of thymol decreased significantly (89-97% of disappearance) in a period of three months, though about 42 µg/kg of thymol persisted in larvae. Thymol was found to accumulate in honey and beeswax at a level of 882 μg/kg and 1305 μg/kg respectively, in the studied period. As the main issue of thymol residues in honey is related to the modification of organoleptic properties, more than toxicology, the levels found in this study indicate that the taste of honey may be affected during and up to three months after the treatment application. In addition, we did not find adverse effects on the evolution of the colonies due to the applied treatment.
Highly effective pre-concentration of thymol and carvacrol using nano-sized magnetic molecularly imprinted polymer based on experimental design optimization and their trace determination in summer savoury, Origanum majorana and Origanum vulgare extracts
2021, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
It has several applications at low concentration (lower than 1 mM) in (a) cosmetic formulations (b) food (as a preservative against food microorganisms and artificial flavor) (c) foreign drugs due to its preservative properties, and (d) insecticides, fungicides and other chemical products [10]. Thyme, savory, oregano, basil, etc., are the most important sources of thymol [11,12]. Sample preparation techniques are required in environmental and food samples for trace detection of target analytes.
To ascertain thymol and carvacrol in pharmaceutical syrups, a valid and effective magnetic molecular imprinted polymer dispersive solid phase microextraction (MMIP-DSPME) process was developed in this study, which was in combination with a high performance liquid chromatography-ultra violet (HPLC-UV) technique for the assessment of thymol and carvacrol separation and pre-concentration. Contact time, eluent kind and volume, pH, the mass of the MMIP were all taken into consideration as key factors. Design expert and multi-objective response surface methodology (RSM) were used to optimize these variables. The mass of the MMIP, sample pH, eluent kind, time of sorption, the volume of eluent, and time of elution were 10 mg, 6, acetonitrile, 28 min, 200 µL, and 5.5 min, respectively, for the maximum extraction recovery of the analytes. The limit of detection (LOD) was 0.042 ng mL⁻¹ at the optimal conditions, while the value for the limit of quantification (LOQ) was 0.140 ng mL⁻¹. At the optimized conditions for thymol and carvacrol, the suggested MMIP sorbent had sorption capacities of 64.1 and 72.6 mg g⁻¹, respectively. Furthermore, for triplicate measurements, the linear dynamic range (LDR) was 0.40–5000 ng mL⁻¹, and the method's accuracy (RSD %) was 6.26%. The saturation magnetization for the MMIP was 19.0 emu g⁻¹ obtained by VSM, allowing the sorbent to be separated quickly. The sorption experiments confirmed the large sorption capacity of the MMIP for thymol and carvacrol, as well as its homogeneous binding sites. The extraction recovery for thymol and carvacrol was 96.9–103.8% and 96.6–105.4%, respectively, at all spiked amounts (20, 100, 200, and 500 ng mL⁻¹). The findings of seven desorption–regeneration cycles using MMIP demonstrated the high stability of the sorbent. The MMIP revealed a particular behavior of sorption for thymol and carvacrol, implying a selective, simple, effective, and flexible analytical method.
Thymol bioactivity: A review focusing on practical applications
2020, Arabian Journal of Chemistry
Citation Excerpt :
For these reasons, the authors suggest suspending treatment for detoxification times for bees, although this could have a lower effect on treatment (Glavan et al., 2020). It is also important to note that traces of thymol have been reported in products related to bees such as honey and pollen but below the threshold that would cause changes in taste and odor (Ares et al., 2020; Tananaki et al., 2014). Similarly, Zolfaghari and Vatanparast (2020) studied the possible toxic effects of thymol on the snail Caucasotachea atrolabiata, evaluating its action on the neuronal activity of the mollusk, and its mechanism of action.
Thymol is a natural volatile monoterpenoid phenol that is the main active ingredient of oil extracted from species Thymus vulgaris L., commonly known as thyme, and other plants such as Ocimum gratissimum L., Origanum L., Carum copticum L., different species of the genus Satureja L., Oliveria decumbens Vent, and many others. It is a versatile molecule with a wide variety of practical applications such as medical, dentistry, veterinary, food, and agrochemicals, among others. Its pharmacological applications have been the most investigated and reported, focusing on its prominent antimicrobial, antioxidant, anti-inflammatory, cicatrizing activities. Furthermore, it is noteworthy that the research on its agricultural applications has increased, highlighting its uses as a natural agrochemical and preservative to safeguard foods from pathogenic microorganisms both in sowing and storage, which could have a beneficial effect on human health and the environment. Research has also been reported on its activity as an insecticide, acaricide, and animal repellent. This review summarizes important aspects of thymol such as its bioavailability, synthesis, and biological activities, with special interest in practical applications.
A Straightforward and Green Electrochemical Approach for the Simultaneous Determination of Thymol and Carvacrol in Essential Oils
2024, SSRN
Electrode Based on the MWCNTs and Electropolymerized Thymolphthalein for the Voltammetric Determination of Total Isopropylmethylphenols in Spices
2023, Micromachines

View all citing articles on Scopus

View full text

Simultaneous determination of carvacrol and thymol in bee pollen by using a simple and efficient solvent extraction method and gas chromatography-mass spectrometry

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Reagents and materials

Optimization of the sample treatment

Conclusions

Funding

CRediT authorship contribution statement

Declaration of Competing Interest

Acknowledgements

J. Pharm. Biomed. Anal.

J. Food Anal.

J. Chromatogr. A

Talanta

Talanta

Sci. Total Environ.

J. Pharm. Biomed. Anal.

LWT- Food Sci. Technol.

J. Chromatogr. A

J. Pharm. Biomed. Anal.

Trends Analyt. Chem.

Biochem. Syst. Ecol.

J. Supercritic. Fluid.

Ind. Crops Prod.

Study of the potential synergistic antibacterial activity of essential oil components using the thiazolyl blue tetrazolium bromide (MTT) assay

LWT-Food Sci. Technol.