Determination of Dicyclohexylamine in Beeswax by Aqueous Normal Phase Liquid Chromatography Coupled With Tandem Mass Spectrometry.
Dicyclohexylamine (DCH) is an excipient present in commercial formulations of fumagillin (Fumagilin-B® and Fumidil-B®), an antibiotic which is employed in apiculture for the control of nosema disease. DCH has been demonstrated to be stable in harvested honey; however, its fate in the beehive has not been investigated. In this study, DCH residues were determined in beeswax samples collected from brood chambers and honey supers from various apiaries throughout Alberta. The determination of DCH was performed using a simple extraction procedure followed by low-temperature cleanup and finally analysis with aqueous normal phase liquid chromatography-electrospray ionization tandem mass spectrometry. The method was found to yield high accuracy and precision (94.9 ± 1.7% to 110.8 ± 6.6%) for replicate beeswax samples spiked with 4, 80, 360 and 2,000 μg kg-1 of DCH. With a limit of quantification of 1 μg kg-1, 100% of the 61 beeswax samples analyzed were found to contain residues of DCH ranging from 15 to 6,410 μg kg-1. The results indicate that DCH can accumulate in beeswax when Fumagilin-B® or Fumidil-B® is applied in apiculture and that these concentrations can vary significantly among beekeeping operations.
In Vivo Inhibitory Assessment of Potential Antifungal Agents on Nosema ceranae Proliferation in Honey Bees
Nosema ceranae Fries, 1996, causes contagious fungal nosemosis disease in managed honey bees, Apis mellifera L. It is associated around the world with winter losses and colony collapse disorder. We used a laboratory in vivo screening assay to test curcumin, fenbendazole, nitrofurazone and ornidazole against N. ceranae in honey bees to identify novel compounds with anti-nosemosis activity compared to the commercially available medication Fumagilin-B®. Over a 20-day period, Nosema-inoculated bees in Plexiglas cages were orally treated with subsequent dilutions of candidate compounds, or Fumagilin-B® at the recommended dose, with three replicates per treatment. Outcomes indicated that fenbendazole suppressed Nosema spore proliferation, resulting in lower spore abundance in live bees (0.36 ± 1.18 million spores per bee) and dead bees (0.03 ± 0.25 million spores per bee), in comparison to Fumagilin-B®-treated live bees (3.21 ± 2.19 million spores per bee) and dead bees (3.5 ± 0.6 million spores per bee). Our findings suggest that Fumagilin-B® at the recommended dose suppressed Nosema. However, it was also likely responsible for killing Nosema-infected bees (24% mortality). Bees treated with fenbendazole experienced a greater survival probability (71%), followed by ornidazole (69%), compared to Nosema-infected non-treated control bees (20%). This research revealed that among screened compounds, fenbendazole, along with ornidazole, has potential effective antifungal activities against N. ceranae in a controlled laboratory environment.
Development of new analytical methods for the determination of caffeine content in aqueous solution of green coffee beans
BackgroundThis study was conducted to develop fast and cost effective methods for the determination of caffeine in green coffee beans. In the present work direct determination of caffeine in aqueous solution of green coffee bean was performed using FT-IR-ATR and fluorescence spectrophotometry. Caffeine was also directly determined in dimethylformamide solution using NIR spectroscopy with univariate calibration technique.ResultsThe percentage of caffeine for the same sample of green coffee beans was determined using the three newly developed methods. The caffeine content of the green coffee beans was found to be 1.52 ± 0.09 (% w/w) using FT-IR-ATR, 1.50 ± 0.14 (% w/w) using NIR and 1.50 ± 0.05 (% w/w) using fluorescence spectroscopy. The means of the three methods were compared by applying one way analysis of variance and at p = 0.05 significance level the means were not significantly different. The percentage of caffeine in the same sample of green coffee bean was also determined by using the literature reported UV/Vis spectrophotometric method for comparison and found to be 1.40 ± 0.02 (% w/w).ConclusionNew simple, rapid and inexpensive methods were developed for direct determination of caffeine content in aqueous solution of green coffee beans using FT-IR-ATR and fluorescence spectrophotometries. NIR spectrophotometry can also be used as alternative choice of caffeine determination using reduced amount of organic solvent (dimethylformamide) and univariate calibration technique. These analytical methods may therefore, be recommended for the rapid, simple, safe and cost effective determination of caffeine in green coffee beans.