Determination of quinclorac and quinclorac methyl ester in honey by online SPE-UPLC-MS/MS.

A method employing online solid phase extraction (SPE) coupled to UPLC-MS/MS was developed for the determination of residues of the acid herbicide quinclorac plus its transformation product, quinclorac methyl ester, in honey. The analytical method involved dissolving the honey in a mixture of methanol:water followed by direct injection into a two-dimensional UPLC system which is used to perform an automated SPE cleanup on a reusable phenyl cartridge prior to the target analytes being transferred onto an analytical UPLC column for subsequent chromatographic separation followed by MS/MS detection. The limits of quantitation for quinclorac and quinclorac methyl ester in honey were both set at 0.5 µg kg-1 and the method detection limit was estimated to be 0.012 µg kg-1 for each compound. The working analytical range (0.5-100 µg kg-1) was validated by analysing a series of spiked replicate honey samples. The method was applied to the analysis of various honeys obtained from numerous different commercial sources. Quinclorac was detected in 9 out of 30 samples at concentrations ranging from 0.6 to 31.5 µg kg-1. Quinclorac methyl ester, which is estimated to be significantly more toxic than the parent herbicide itself, was not detected in any honey sample.

Tylosin A and desmycosin in honey by salting-out assisted liquid-liquid extraction and aqueous normal phase ultraperformance liquid chromatography-tandem mass spectrometry.

A simple and rapid method was developed for the determination of tylosin A and desmycosin residues in honey. Aliquots of honey samples were dissolved in a concentrated solution of sodium acetate and the target analytes were subsequently extracted with acetonitrile. The resulting organic extract was chromatographed under aqueous normal phase (ANP) LC conditions using a bare silica stationary phase with acidified solutions of ammonium formate in both water and 5:95 water: acetonitrile as the mobile phases. Tylosin A and desmycosin residues were measured using MS/MS in the multiple reaction monitoring (MRM) mode. Based on the analysis of replicate honey samples fortified at 5, 20, and 100 µg kg-1, the method was found to provide high accuracy and precision with average intraday trueness ranging from 90.2 to 111.2% and standard deviations of less than 7%. For spiked replicates fortified at the limit of quantification (1 µg kg-1), the intraday accuracies ranged from 72.0 to 102.7% for tylosin A and from 72.1 to 93.8% for desmycosin, with standard deviations all lower than 12%. Matrix effects were relatively minimal and consistent between honey samples which eliminated the need to perform any additional cleanup of the sample extracts prior to ANP-UPLC-MS/MS analysis. Graphical abstract.

Determination of glyphosate, AMPA, and glufosinate in honey by online solid-phase extraction-liquid chromatography-tandem mass spectrometry.

A simple method was developed for the simultaneous determination of glyphosate, its main degradation product (aminomethylphosphonic acid), and glufosinate in honey. Aqueous honey solutions were derivatised offline prior to direct analysis of the target analytes using online solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry. Using the developed procedure, accuracies ranging from 95.2% to 105.3% were observed for all analytes at fortification levels of 5, 50, and 150 µg kg-1 with intra-day precisions ranging from 1.6% to 7.2%. The limit of quantitation (LOQ) was 1 µg kg-1 for each analyte. Two hundred honey samples were analysed for the three analytes with AMPA and glyphosate being most frequently detected (99.0% and 98.5% of samples tested, respectively). The concentrations of glyphosate were found to range from <1 to 49.8 µg kg-1 while those of its degradation product ranged from <1 to 50.1 µg kg-1. The ratio of glyphosate to AMPA was found to vary significantly amongst the samples where both analytes were present above the LOQ. Glufosinate was detected in 125 of 200 samples up to a maximum concentration of 33.0 µg kg-1.

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.

Stability of dicyclohexylamine and fumagillin in honey.

Fumagillin is extensively used to control nosema disease in apiculture. In the commercial formulation, fumagillin is present as a salt in an equimolar quantity with dicyclohexylamine (DCH). In this study DCH was observed to be significantly more resistant to degradation in honey than fumagillin using LC-MS/MS analysis. Observed half-lives for DCH ranged from a minimum of 368 days when kept at 34 °C in darkness, to a maximum of 852 days when stored at 21 °C in darkness. A maximum half-life of 246 days was observed for fumagillin in samples kept in darkness at a temperature of 21 °C. The observed half-life of fumagillin was estimated to be 3 days when exposed to light at 21 °C, and complete decomposition was observed after 30 days under the same conditions. The stability of DCH, combined with its genotoxicity and tumorigenic properties make it an important potential contaminant in honey destined for human consumption.

Fumagillin: an overview of recent scientific advances and their significance for apiculture.

Fumagillin is a potent fungal metabolite first isolated from Aspergillus fumigatus. It is widely used in apiculture and human medicine against a variety of microsporidian fungal infections. It has been the subject of research in cancer treatments by employing its angiogenesis inhibitory properties. The toxicity of fumagillin has limited its use for human applications and spurred the development of analogues using structure-activity relationships relating to its angiogenesis properties. These discoveries may hold the key to the development of alternative chemical treatments for use in apiculture. The toxicity of fumagillin to humans is important for beekeeping, because any residues remaining in hive products pose a direct risk to the consumer. The analytical methods published to date measure fumagillin and its decomposition products but overlook the dicyclohexylamine counterion of the salt form widely used in apiculture.

Degradation of incurred tylosin to desmycosin--implications for residue analysis of honey.

As a result of the application of tylosin to honey bee colonies for the control of American foulbrood disease, antibiotic residues may exist in honey destined for human consumption. It has been recognized that the parent compound, tylosin A, degrades in acidic media such as honey to yield the antimicrobially active degradation product, desmycosin. Data is presented documenting levels of incurred tylosin and desmycosin in honey resulting from simulated therapeutic applications of a commercial formulation of tylosin during the fall. It is demonstrated that honey destined for human consumption should be analyzed for both tylosin A and desmycosin (tylosin B) rather than the parent antibiotic alone. An analytical method that permits the simultaneous determination of tylosin A and desmycosin in honey using liquid chromatography-tandem mass spectrometry is also presented.