SOJ Pharmacy & Pharmaceutical Sciences

A QuEChERS-based extraction protocol combined with a fast and selective UHPLC-QTOF-MS assay for the detection and quantification of destruxins from honey is described. The internal standard fortified assay showed satisfying assay precision and accuracy and allowed quantification of destruxin A and B down to ppb range with limits of detection (LOD) and quantification (LOQ) of less than 1.0 ppb and 2.3 ppb, respectively. Destruxin E showed a distinctive loss of recovery and was excluded from further quantitative analysis. Honey samples obtained from a field trial using Metarhizium brunneum against the ectoparasitic honey bee mite Varroa destructor were examined for possible destruxin residues. No analytes could be detected and samples were found to be residue-free.
Key words: Metarhizium; QuEChER; Honey; Varroa destructor; UHPLC-DAD-QTOF-MS; Risk assessment

Varroa destructor Anderson and Trueman (2000) is an ectoparasitic mite which is currently one of the most serious threats for apiculture [1, 2]. It was originally confined to the Eastern honey bee Apis cerana however it is nowadays a new parasite of the honey bee Apis mellifer a spread almost worldwide within a short time [3]. Since the honey bee A. mellifera is crucial not only for honey production but also for crop pollination varroa control is of great interest [1]. Different approaches to control Varroa destructor have been tried in the last years using mainly synthetic acaricides or organic acids. However, the harmlessness of those substances was questioned the development of alternative treatments is of great necessity [1, 2]. A promising alternative strategy could therefore be the use of entomopathogenic fungi e.g. Metarhizium spp. or Beauveria spp [3, 4].
Although M. brunneum plays an important role as a biological control agent (BCA) [5] there are still concerns that its bioactive secondary metabolites, so called destruxins (dtxs), imparts risks to humans and the environment. Hence, the availability of analytical assays for destruxin trace analysis is of great necessity in order to answer the question whether destruxins might be able to enter the food chain [6-9].
In this study samples from bee hives treated with M. brunneum strain BIPESCO 5 (listed in regulation EU No. 540/2011 [10]) were examined for possible metabolite residues. Within the following paragraphs the extension of the developed UHPLCQTOF- MS method [7] for the detection and quantification of destruxins from honey samples is presented.

Destruxin reference materials (dtxA, dtxB, dtxE, NMR purity greater than 98%) were isolated from M. brunneum strain BIPESCO 5 as previously described [11]. Acetonitrile (gradient grade), was purchased from Merck (Darmstadt, Germany); ammonium formate (for mass spectrometry ≥ 99.0%) and roxithromycin (HPLC grade ≥90%) were purchased from Sigma Aldrich (Vienna, Austria). Nitrogen for mass spectrometry was produced on site by a nitrogen generator (Peak Scientific Instruments, Fountain Crescent, UK). Water for the HPLC was produced on site (Sartorius stedim, arium 611UV, Sartorius, Vienna, Austria).

Always two stock solutions containing either a combination of dtxA, dtxB, and dtxE or the internal standard roxithromycin (IS) (fig. 1) in acetonitrile were prepared. In all cases sample volumes were 1 ml. Starting from these stock solutions a series of eight spike solutions containing dtxs and the IS were prepared.

with following concentrations: 0.05, 0.125, 0.25, 0.375, 0.5, 1.25, 2.5, 5.0 μg/ml dtxs and the internal standard at a concentration of 0.5 μg/ml. Obtained spike solutions were then used for the preparation of final calibration levels in either acetonitrile by a 1:50 dilution or by spiking 100 μl into 5 g honey sample. A third stock solution containing dtxA, dtxB, dtxE was used to generate quality control samples (QC) for the assessment of precision and accuracy. Spike solutions fortified with different concentrations of the analytes (0.25, 0.5, 2.5 and 3.75 μg/ml) and identical amounts of the IS (0.5 μg/ml) were prepared and final QC samples obtained by spiking 100 μl into honey sample.

Sample preparation was carried out based on the protocol described by Taibon et al. [7]. Samples for calibration curves and quality control samples were spiked with the appropriate amounts of analytes (dtxA, dtxB and dtxE) and the internal standard roxithromycin (0.5 μg/ml), while real samples for destruxin quantification were spiked only with the IS solution (0.5 μg/ml).
Five g (± 0.1 g) of honey samples were weighed into an extraction tube, mixed with 10 ml water and shaken until the honey was completely dissolved. Extraction was carried out with 10 ml of acetonitrile (ACN) followed by dispersive solid phase extraction (d-SPE) for final purification. An aliquot of 1 ml of the purified sample was than evaporated to dryness and re-dissolved in 500 μl of acetonitrile prior to analysis.

An Agilent 1200 UHPLC liquid chromatography (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector (DAD), an autosampler set at 10°C, an automatic injector, and a column oven set at 40°C was used for all measurements [7]. Separations were performed on a Zorbax Eclipse XDB-C18 rapid resolution column (50 x 2.1 mm, 1.8 μm particle size, Agilent) guarded with an in-line filter (0.2 μm pore size frit, 2.1 mm diameter, Agilent) with water (A) and 95% acetonitrile (v/v) (B), each containing 10 mM ammonium formate, serving as mobile phase. The system was operated at a flow rate of 0.3 ml/min with following gradient: t= 0 min 75% A, t= 0.5 min 75% A, t= 3.5 min 60% A, t= 4.5 min 50% A, t= 5.5 min 35% A, t= 6.5 min 5% A, t= 7.0 min 2% A, t= 12 min 2% A. Between runs the column was equilibrated with 75% A for 12 min. The injection volume was 2.5 μl and chromatograms were recorded at 210 nm.
For destruxin quantification the UHPLC-DAD system was hyphenated to a Bruker micrOTOF-QII mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in positive ESImode. All MS experiments were performed with the ionizer and transfer parameters as described [7].

Calibration functions were obtained for a dilution series in acetonitrile and in honey serving as matrix model. Spiked matrix samples were prepared using the QuEChERS protocol described above, whereas calibration levels in acetonitrile were directly analysed without further treatment. All calibration levels were measured in triplicates and calibration functions generated using analysis software QuantAnalysis (Version 2.0 SP4, Bruker). For both calibration models the limit of detection (LOD) and the limit of quantification (LOQ) were calculated from calibration functions including only the lowest four fortification levels. From the obtained regression equation data LOD was calculated as three times the standard deviation (STD) of the y-intercept divided by the slope, whereas LOQ was calculated as ten times the STD of the y-intercept divided by the slope [7,12,13].
Honey samples, fortified with four different concentrations of analytes and identical amounts of IS, were used to asses precision and accuracy of the assay. For intra- and inter-day repeatability samples were measured on three different days in triplicate. Precision was determined as the relative standard deviation (RSD) of the respective replicate measurements of the different levels; accuracy was expressed as percentage of recovery of the measured analyte concentration in relation to the spiked concentration.

Honey samples obtained from beehives treated with M.brunneum (BIPESCO 5) were used for the screening of fungal metabolites [14]. In total 16 beehives, eight for the treatment with BIPESCO5 and eight for control were used. M. brunneum treatment occurred from June to July once a week in four repetitions dusting every frame on both sides with10 g of conidial spores per hive (concentration 1010CFU/g). For the screening of destruxins honeycombs of every hive filled with honey were taken and stored at 8°C until further use.

Assay validation was performed with dilution series containing dtxA, dtxB, dtxE and the IS roxithromycin in acetonitrile and honey serving as matrix model. While spiked matrix samples had to be prepared using the described QuEChERS protocol, calibration levels in acetonitrile were directly analysed. Final analyte concentrations in ng/ml in acetonitrile corresponded to ppb in matrix samples. All calibration levels were measured in triplicates and results evaluated using the analysis software. Calibration curves were obtained for all three analytes in both matrix models resulting in quadratic functions with correlation coefficient values (R²) between 0.9992 and 0.9999. Calibration ranges for both dilution series in acetonitrile and honey ranged from 1.0 – 100.0 ng/ml (ppb) for dtxA and dtxB and from 2.5 – 100.0 ng/ml (ppb) for dtxE. Independent of the matrix model used for calculation limits of detection (LOD) and limits of quantification (LOQ) for dtxs A and B were found to be less than 1 ng/ml (ppb) and 2.3 ng/ml (ppb), respectively. Destruxin E showed comparable values for the dilution series in acetonitrile, but much higher results for the dilution series in honey with a LOD of 3.7 ng/ml(ppb) and a LOQ of 11.3 ng/ml (ppb). The analytical sensitivity (slope of the calibration function) in reference solutions in acetonitrile was identical to honey matrix samples for dtxA and dtxB but showed a lower value for the calibration curve of dtxE in honey, which indicated a loss of the respective metabolite (tab. 1).
Assay precision and accuracy was assessed using spiked matrix samples at four different concentrations each prepared in triplicates. For determination of intra-day and inter-day precision all levels were measured three times on three different days. For all fortification levels of dtxA and dtxB intra-day RSD ranged between 4.9% and 14.9% and inter-day RDS was found to be less than 12.6%. Assay accuracy for dtxA and dtxB ranged between 83.4% and 115.0% independent on the calibration curve used for calculation (tab. 2). As expected form the differences in the slope of the calibration function for dtxE in acetonitrile and honey matrix, spike experiments confirmed the loss of the respective analyte (Fig. 2). This data are comparable with results obtained in a previously performed study for strawberry and maize proving the assay matrix independent with the exception of dtxE in maize, where also an analyte loss was observed [7]. Consequently quantification of destruxin congeners in honey has to be limited to dtxA and dtxB, which are known as one of the major metabolites secreted by the investigated M. brunneum strains [6, 11, 13, 15, 16]. This allows the use of respective destruxins as surrogate markers for the presence of M. brunneum or its secondary metabolites in honey samples.

The developed assay was used to screen honey samples obtained from a trial where the fungus Metarhizium brunneum, strain BIPESCO 5, was used to control the ectoparasitic mite Varroa destructor in honey bee colonies. From the original sixteen samples seven controls and seven treatment samples were analysed. All samples were prepared in duplicate and measured three times. No traces of destruxins were found in any of the analysed samples.

With this study the applicability of the developed UHPLCQTOF- MS method has been extended to the analysis of destruxins in honey. The combination of the QuEChERS based sample preparation protocol with the chromatographic system hyphenated to a high resolution TOF mass spectrometer (UHPLCQTOF- MS) allowed quantification of dtxA and dtxB down to ppb range. Overall, the assay showed satisfying sensitivity, precision and accuracy and can be considered as stable and reproducible. The suitability of the assay was further proved by analysing real samples from a field study using M. brunneum to control the ectoparasitic mite Varroa destructor in bee colonies. No traces of destruxins were found in any of the analysed samples.
Monitoring pesticide residues in honey is of great necessity not only for synthetic pesticides but also for biological pest control agents and can help to assess their potential risks to consumer health and environment. Consequently this assay can be considered as a routine method for destruxin trace analysis in honey samples.

This research has been supported by the European Community’s Seventh Framework Programme grant (FP7_ ENV.2011.3.1.9-1 ECO-INNOVATION, INBIOSOIL, Grant Agreement No. 282767).

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