The honey bee (
Depletion of residues of pharmacologically active substances in honey is not time-dependent, in contrast to their depletion in mammalian or avian organisms due to pharmacokinetic behaviour. When the residues are found in honey, they mostly stay there (5). Because of this, using sulphonamides illegally may cause their residue accumulation in honey and other apiculture products like beeswax (7, 10). Apart from honey, beeswax is a valuable beehive product. It is used by the food, cosmetic and pharmaceutical industries in a wide range of applications that require high-quality beeswax. The product is classified as an authorised food additive (E 901) in the EU and is listed in the European Pharmacopoeia (2). Since it is a natural product, there should not be any additives in it.
However, beeswax can dissolve or integrate toxic compounds, which could be released long afterwards when the beeswax is used to produce pharmaceuticals or cosmetics, is eaten, or is given to honey bees as a wax foundation. Previous work has focused only on sulphamethazine, which can contaminate honey during the next season if it stays in the comb’s wax after being used in the hive (11). To the best of our knowledge, no similar studies have been carried out for other sulphonamides. Therefore, the aim of the research was to compare the migration of 10 sulphonamides from contaminated beeswax to honey.
All solvents and chemical compounds used were of analytical or liquid chromatography grade. JT Baker (Deventer, the Netherlands) provided acetonitrile, acetic acid, ammonium hydroxide solution, isopropanol, methanol and n-hexane. A Milli-Q plus water purification system from Millipore (Bedford, MA, USA) produced ultrapure water. Strata SCX solid-phase extraction (SPE) tubes (500 mg, 3 mL) were supplied by Phenomenex (Torrance, CA, USA). The PVDF syringe filters (0.45 μm, 13 mm) were provided by Restek (Bellefonte, PA, USA). High purity analytical standards (>98.5%) of sulphadimethoxine (SDM), sulphadoxine (SDX), sulphamonomethoxine (SMM), sulphamethoxazole (SMX), sulphameter (SMT), sulphamethazine (SMZ), sulphamerazine (SMR), sulphadiazine (SDA), sulphathiazole (STZ) and sulphacetamide (SCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sulphadimethoxine-D6 (SDM-D6), sulphadoxine-D3 (SDX-D3), sulphamonomethoxine-13C6 (SMM-13C6), sulphamethoxazole-13C6 (SMX-13C), sulphamethazine-13C6 hemihydrate (SMZ-13C6), sulphamerazine-13C6 (SMR-13C6), sulphadiazine-13C6 (SDA-13C6) and sulphathiazole-13C6 (STZ-13C6) were obtained from Witega Laboratorien Berlin-Adlershof (Berlin, Germany), and sulphameter-D4 (SMT-D4) was acquired from Toronto Research Chemicals (Toronto, ON, Canada). Isotopically labelled analytes used as internal standards (ISs) were of chemical and isotopic purity greater than 98%.
Blank beeswax was melted at 80°C, and a mixture of 10 sulphonamides in methanol was added to produce wax foundations (n = 4) containing sulphonamides at a concentration of 10,000 μg/kg. After mixing, liquid sulphonamide-containing beeswax was poured into a wax foundation mould and allowed to cool down. Next, a small portion of every beeswax foundation was removed for liquid chromatography−tandem mass spectrometry (LC-MS/MS) sulphonamide analysis. Subsequently, the sulphonamide-contaminated wax foundations were put into wooden frames (260 × 360 × 35 mm). In mid-June, when it was the summer flowering season, each frame was placed near the brood nest in a separate hive so that the honey bees (
Stock solutions of individual analytical standards (1,000 μg/mL) were prepared in acetonitrile (stable at −20°C for at least a year). Next, the solutions were combined and diluted with 0.1% acetic acid to create sulphonamide and IS working standard solutions, which, when not in use, were kept in amber glass at 4°C where they were stable for at least six months.
Honey analysis was performed as previously reported by Mitrowska
An LCMS-8050 triple-quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) with an electrospray ionisation source and Lab Solutions LCMS 5.60 SP2 software (Shimadzu) was used to analyse honey and beeswax extracts. Sulphonamides were separated chromatographically in gradient mode on a Luna analytical column of 150 × 2 mm with 3 μm particle size, in which the stationary phase was pentafluorophenyl (Phenomenex). Mobile phase A contained 0.01% acetic acid in acetonitrile, and mobile phase B contained 0.01% acetic acid in water. More information about LC-MS/MS conditions and ion transitions monitored can be found in Mitrowska
Spiked and blank quality control honey and beeswax samples were analysed with each sample series. The sulphonamide concentrations in honey and beeswax samples were determined by comparing the ratio of a sulphonamide peak area to its corresponding IS peak area with the same ratio in the calibration curves (2–200 μg/kg for honey and 400–40,000 μg/kg for beeswax).
The significance of the differences was determined using analysis of variance.
The methods used to quantify sulphonamides in honey and beeswax were validated by determining specificity, linearity, intermediate precision, recovery, limit of detection and limit of quantification. The specificity of the methods was assessed by examining 20 blank honey and beeswax samples. During the retention time of the target compounds, no interfering peaks from natural substances were observed. The calibration curves demonstrated high linearity for each analyte in the concentration range of 2–200 μg/kg for honey and 400–40,000 μg/kg for beeswax (correlation coefficient > 0.99). For each sulphonamide in honey, the limit of detection and limit of quantification were 1 and 2 μg/kg, respectively, whereas the corresponding values in beeswax were 200 and 400 μg/kg. The recoveries of sulphonamides from honey (at 2, 25 and 100 μg/kg) and from beeswax (at 1,000, 5,000 and 10,000 μg/kg) ranged from 68.1 to 99.9% with a coefficient of variation < 16.6% under intermediate precision conditions (Table 1). The data showed that the methods for quantification of sulphonamides in honey and beeswax were accurate, precise and fit for use in these studies.
Validation parameters calculated for determination of sulphonamides in honey and beeswax samples (n = 18)
Honey | Beeswax | |||||
---|---|---|---|---|---|---|
Sample | Concentration (μg/kg) | Recovery (%) | Intermediate precision (CV, %) | Concentration (μg/kg) | Recovery (%) | Intermediate precision (CV, %) |
SCA | 2 | 69.7 | 15.1 | 1,000 | 71.9 | 12.7 |
25 | 68.1 | 14.9 | 5,000 | 73.1 | 14.6 | |
100 | 72.4 | 16.6 | 10,000 | 69.1 | 13.2 | |
SDA | 2 | 96.7 | 12.9 | 1,000 | 98.2 | 11.4 |
25 | 98.4 | 11.7 | 5,000 | 99.2 | 12.3 | |
100 | 99.3 | 13.2 | 10,000 | 97.7 | 12.9 | |
STZ | 2 | 93.3 | 13.4 | 1,000 | 98.9 | 10.4 |
25 | 93.2 | 11.8 | 5,000 | 99.1 | 12.3 | |
100 | 94.2 | 14.2 | 10,000 | 97.2 | 10.9 | |
SMR | 2 | 97.2 | 14.8 | 1,000 | 97.4 | 10.1 |
25 | 99.7 | 13.2 | 5,000 | 98.6 | 10.9 | |
200 | 98.3 | 14.6 | 10,000 | 97.7 | 12.3 | |
SMZ | 2 | 97.4 | 11.9 | 1,000 | 99.1 | 12.9 |
25 | 99.7 | 13.2 | 5,000 | 98.6 | 10.4 | |
200 | 98.1 | 12.8 | 10,000 | 99.7 | 12.1 | |
SMT | 2 | 98.0 | 12.3 | 1,000 | 97.1 | 11.7 |
25 | 96.7 | 11.1 | 5,000 | 98.5 | 13.2 | |
200 | 99.3 | 12.8 | 10,000 | 97.9 | 10.5 | |
SMM | 2 | 96.9 | 13.7 | 1,000 | 99.9 | 12.1 |
25 | 98.0 | 12.9 | 5,000 | 97.0 | 10.3 | |
200 | 99.6 | 14.1 | 10,000 | 99.1 | 12.2 | |
SDX | 2 | 97.0 | 10.4 | 1,000 | 98.2 | 13.2 |
25 | 99.9 | 11.2 | 5,000 | 97.9 | 11.5 | |
200 | 98.3 | 10.7 | 10,000 | 99.7 | 13.3 | |
SMX | 2 | 96.1 | 14.5 | 1,000 | 98.0 | 11.8 |
25 | 97.8 | 13.8 | 5,000 | 96.6 | 11.9 | |
200 | 99.7 | 13.2 | 10,000 | 98.8 | 12.8 | |
SDM | 2 | 95.6 | 15.9 | 1,000 | 96.3 | 12.9 |
25 | 94.2 | 13.2 | 5,000 | 99.3 | 12.6 | |
200 | 97.8 | 15.2 | 10,000 | 97.0 | 10.2 |
CV – coefficient of variation; SCA – sulphacetamide; SDA – sulphadiazine; STZ – sulphathiazole; SMR – sulphamerazine; SMZ – sulphamethazine; SMT – sulphameter; SMM – sulphamonomethoxine; SDX – sulphadoxine; SMX – sulphamethoxazole; SDM – sulphadimethoxine
The results of the sulphonamide analysis of wax foundations made from beeswax fortified with 10 sulfonamides at 10,000 μg/kg revealed that some quantity of the added substances had been lost as a result of thermal degradation occurring when the wax was heated to 80°C. In these conditions, the sulphonamides were stable in a range from 20 to 53%. The mean concentrations of SDM, SMM, SDX, SMX, SMZ, SMR, SDA, SMT, SCA and STZ present in the wax foundation were 4,625, 5,326, 4,541, 3,824, 4,138, 4,358, 5,027, 1,993, 4,409 and 3,072 μg/kg, respectively (Table 2).
The concentrations and recoveries of sulphonamides calculated for the beeswax foundation fortified with 10 sulphonamides at 10,000 μg/kg (n = 4)
Analyte | Concentration (μg/kg) | Recovery (%) | Analyte | Concentration (μg/kg) | Recovery (%) |
---|---|---|---|---|---|
SDM | 4,625 ± 287 | 46 | SMR | 4,358 ± 260 | 44 |
SMM | 5,326 ± 399 | 53 | SDA | 5,027 ± 318 | 50 |
SDX | 4,541 ± 383 | 45 | SMT | 1,993 ± 156 | 20 |
SMX | 3,824 ± 406 | 38 | SCA | 4,409 ± 370 | 44 |
SMZ | 4,138 ± 152 | 41 | STZ | 3,072 ± 236 | 31 |
SDM – sulphadimethoxine; SMM – sulphamonomethoxine; SDX – sulphadoxine; SMX – sulphamethoxazole; SMZ – sulphamethazine; SMR – sulphamerazine; SDA – sulphadiazine; SMT – sulphameter; SCA – sulphacetamide; STZ – sulphathiazole
The results indicated that each sulphonamide could be transferred from contaminated beeswax to honey. The highest concentrations of SDM (87.2 μg/kg), SMM (74.6 μg/kg), SDX (68.9 μg/kg), SMX (50.9 μg/kg), SMZ (46.0 μg/kg), SMR (36.0 μg/kg), SDA (35.8 μg/kg), SMT (26.0 μg/kg), SCA (16.7 μg/kg) and STZ (12.8 μg/kg) were found in honey from the comb built of the contaminated wax-based foundations fortified at 10,000 μg/kg four months from the beginning of the experiment (Fig. 1). The frames used in the study were completely filled with honey on both sides of the comb. Thus, the ratio of honey (2,448 g) to beeswax (110 g) at each sampling point on the comb was always 22.25 : 1. Applying this proportionality, the maximum transfers of the initial actual amount of SDM, SDX, SMM, SMX, SMT, SMZ, SMR, SDA, STZ and SCA from sulphonamide-containing wax-based foundations to honey were 42.6, 34.3, 31.7, 30.1, 29.5, 25.2, 18.7, 16.1, 9.5 and 8.6%, respectively (Table 3).
The maximum transfer (%) of sulphonamides from tainted beeswax to the honey stored in the comb
Analyte | The maximum transfer (%) of sulphonamides | |||||
---|---|---|---|---|---|---|
1 month | 2 months | 3 months | 4 months | 5 months | 6 months | |
SDM | 13.2 | 15.7 | 21.1 | 42.6 | 20.4 | 9.9 |
SDX | 10.3 | 13.4 | 17.9 | 34.3 | 18.3 | 8.1 |
SMM | 10.0 | 11.5 | 15.8 | 31.7 | 14.9 | 6.9 |
SMX | 7.9 | 9.3 | 13.9 | 30.1 | 13.5 | 5.1 |
SMT | 8.4 | 12.3 | 17.7 | 29.5 | 18.6 | 9.0 |
SMZ | 8.2 | 9.6 | 13.6 | 25.2 | 12.3 | 5.8 |
SMR | 6.2 | 7.0 | 10.6 | 18.7 | 9.7 | 4.2 |
SDA | 4.5 | 5.6 | 8.6 | 16.1 | 8.4 | 3.9 |
STZ | 2.2 | 3.2 | 5.6 | 9.5 | 6.4 | 2.3 |
SCA | 3.6 | 5.0 | 7.8 | 8.6 | 8.5 | 4.0 |
SDM – sulphadimethoxine; SDX – sulphadoxine; SMM – sulphamonomethoxine; SMX – sulphamethoxazole; SMT – sulphameter; SMZ – sulphamethazine; SMR – sulphamerazine; SDA – sulphadiazine; STZ – sulphathiazole; SCA – sulphacetamide. Combs were completely filled with honey on both sides, thus the ratio of honey (2,448 g) to beeswax (110 g) at each sampling point on the comb was always 22.25 : 1
Sulphonamides were not detected in the honey samples taken from negative control honeycombs in experimental hives or other hives in the same apiary, indicating that the contaminated beeswax was the source of all sulphonamides observed in the honey samples.
In order to have comparable results, the sulphonamide-tainted wax foundations were prepared in the same way as drug-containing wax foundations used in similar experiments (10, 12) by adding analytes at 10,000 μg/kg to blank beeswax melted at 80°C. Although the melting point for beeswax is 62–64°C, increasing the temperature to 80°C was necessary to contaminate the beeswax homogeneously without subjecting the added substances to high temperatures over an extended period. As it transpired, some portions of the added sulphonamides were lost nevertheless, because of thermal degradation. Sulphamonomethoxine was the most stable sulphonamide and degraded 47%, while SMT was revealed to be the least stable, with a loss of 80%. Our results are in line with those obtained by Reybroeck
The analysis of sulphonamides in honey indicated that all the tested compounds could be transferred from contaminated beeswax to honey. During the experiment, the sulphonamide levels in honey appeared to be relatively stable despite their quantities declining subsequent to the four-month point after reaching earlier maximum concentrations. The observed decrease in sulphonamide concentrations after four months could be attributed to the possible degradation of compounds in both honey and beeswax. The stability data obtained by Posyniak
According to Martinello
Predicted pKa and lipophilicity of sulphonamides
Analyte | pKa1 |
% particles in the unionised form at pH 4.2 | log |
Analyte | pKa1 |
% particles in the unionised form at pH 4.2 | log |
---|---|---|---|---|---|---|---|
SDM | 1.95 |
99.3 | 1.26 | SMZ | 2.00 |
99.2 | 0.65 |
SDX | 2.11 |
98.0 | 0.58 | SMR | 2.00 |
99.2 | 0.52 |
SMM | 2.17 |
99.0 | 0.74 | SDA | 2.01 |
99.2 | 0.39 |
SMX | 1.97 |
97.3 | 0.79 | STZ | 2.04 |
96.4 | 0.98 |
SMT | 1.98 |
99.3 | 0.23 | SCA | 2.14 |
95.3 | -0.26 |
– strongest basic;
– strongest acidic; SDM – sulphadimethoxine;
SDX – sulphadoxine; SMM – sulphamonomethoxine; SMX – sulphamethoxazole; SMT – sulphameter; SMZ – sulphamethazine; SMR – sulphamerazine; SDA – sulphadiazine; STZ – sulphathiazole; SCA – sulphacetamide
Because residues of sulphonamides, as well as other veterinary substances and plant protection products, could be transferred from contaminated wax combs to stored honey and pose a health risk to consumers, Wilmart
This investigation’s findings lead us to conclude that every tested sulphonamide can migrate from beeswax in combs to honey stored in those combs, with the highest migration potential for SDM and the lowest for SCA. Consequently, honey contamination may occur through the use of sulphonamide-contaminated beeswax in wax foundations. Therefore, sulphonamides need to be monitored in this matrix to guarantee the high safety and quality of beeswax as a product, beeswax as comb foundation, and honey.