Occurrence of Neonicotinoids in Chinese Apiculture and a Corresponding Risk Exposure Assessment.

Neonicotinoids are the most widely used insecticides worldwide, but there is mounting evidence demonstrating that they have adverse effects on nontarget organisms. However, little is known about the extent of environmental neonicotinoids contamination in China. In this study, a total of 693 honey samples from across China, from both Apis melifera and Apis cerana, were analyzed to examine neonicotinoid concentrations and their geographical distribution, and correlation with the primary plant species from which the honey was obtained. Furthermore, chronic and acute exposure risk and risk ranking for humans eating honey were investigated, and risks to bees were also considered. The results revealed that 40.8% of the samples contained at least one of the five neonicotinoids tested. Honeys from commercial crops were found to be more frequently contaminated with neonicotinoids than those from noncommercial crops. Honey samples from Apis mellifera were more frequently contaminated than those from Apis cerana. The concentrations of neonicotinoids found in honey overlapped with those that have been found to have significant adverse effects on honeybee health. The dietary risk assessments indicated that the levels of neonicotinoids detected in honey were likely to be safe for human consumption.

Unraveling the Metabolic Routes of Retapamulin: Insights into Drug Development of Pleuromutilins.

Retapamulin, a semisynthetic pleuromutilin derivative, is exclusively used for the topical short-term medication of impetigo and staphylococcal infections. In the present study, we report that retapamulin is adequately and rapidly metabolized in vitro via various metabolic pathways, such as hydroxylation, including mono-, di-, and trihydroxylation, and demethylation. Like tiamulin and valnemulin, the major metabolic routes of retapamulin were hydroxylation at the 2ß and 8α positions of the mutilin moiety. Moreover, in vivo metabolism concurred with the results of the in vitro assays. Additionally, we observed significant interspecies differences in the metabolism of retapamulin. Until now, modifying the side chain was the mainstream method for new drug discovery of the pleuromutilins. This approach, however, could not resolve the low bioavailability and short efficacy of the drugs. Considering the rapid metabolism of the pleuromutilins mediated by cytochrome P450 enzymes, we propose that blocking the active metabolic site (C-2 and C-8 motif) or administering the drug in combination with cytochrome P450 enzyme inhibitors is a promising pathway in the development of novel pleuromutilin drugs with slow metabolism and long efficacy.

Deglucosylation of zearalenone-14-glucoside in animals and human liver leads to underestimation of exposure to zearalenone in humans.

Zearalenone-14-glucoside (ZEN-14G), the modified mycotoxin of zearalenone (ZEN), has attracted considerable attention due to its high potential to be hydrolyzed into ZEN, which would exert toxicity. It has been confirmed that the microflora could metabolize ZEN-14G to ZEN. However, the metabolic profile of ZEN-14G and whether it could be deglucosidated in the liver are unknown. To thoroughly investigate the metabolism of ZEN-14G, in vitro metabolism including phase I and phase II metabolism was studied using liquid chromatography coupled to high-resolution mass spectrometry. Additionally, in vivo metabolism of ZEN-14G was conducted in model animals, rats, by oral administration. As a result, 29 phase I metabolites and 6 phase II metabolites were identified and significant inter-species metabolic differences were observed as well. What is more, ZEN-14G could be considerably deglucosidated into its free form of ZEN after the incubation with animals and human liver microsomes in the absence of NADPH, which was mainly metabolized by human carboxylesterase CES-I and II. Furthermore, results showed that the major metabolic pathways of ZEN-14G were deglucosylation, hydroxylation, hydrogenation and glucuronidation. Although interspecies differences in the biotransformation of ZEN-14G were observed, ZEN, α-ZEL-14G, ß-ZEL-14G, α-ZEL, ZEN-14G-16GlcA and ZEN-14GlcA were the major metabolites of ZEN-14G. Additionally, a larger yield of 6-OH-ZEN-14G and 8-OH-ZEN-14G was also observed in human liver microsomes. The obtained data would be of great importance for the safety assessment of modified mycotoxin, ZEN-14G, and provide another perspective for risk assessment of mycotoxin.

[Enzymatic characteristics of peroxidase from Chrysanthemum morifolium cv. Bo-ju].

The enzymatic browning is one of the main reasons for affecting the quality of medicinal flowers. In the process of chrysanthemum harvesting and processing, improper treatment will lead to the browning and severely impact the appearance and quality of chrysanthemum. Peroxidase enzyme is one of the oxidoreductases that cause enzymatic browning of fresh chrysanthemum. The enzymatic characteristics of peroxidase (POD) in chrysanthemum were studied in this paper. In this experiment, the effects of different reaction substrates and their concentrations, PH value of buffer and reaction temperatures on the activity of POD enzyme were investigated. The results showed that the optimal substrate of POD was guaiacol, and the optimal concentration of POD was 50 mmol·L⁻¹. The optimal pH value and reaction temperature were 4.4 and 30-35 °C, respectively. Michaelis-Menten equation was obtained to express the kinetics of enzyme-catalyzed reaction of POD, Km=0.193 mol·L⁻¹, Vmax=0.329 D·min⁻¹. In addition, the results of POD enzyme thermal stability test showed that the POD enzyme activity was inhibited when being treated at 80 °C for 4 min or at 100 °C for 2 min. The above results were of practical significance to reveal the enzymatic browning mechanism, control the enzymatic browning and improve the quality of chrysanthemum, and can also provide the basis for the harvesting and processing of medicinal materials containing polyphenols.

Characterization of Chinese Unifloral Honeys Based on Proline and Phenolic Content as Markers of Botanical Origin, Using Multivariate Analysis.

The phenolic and proline content were determined in honey samples of different floral origins (rapeseed, sunflower, buckwheat and Codonopsis) from five different regions of China. The phenolic and proline profile of these samples were used to construct a statistical model to distinguish honeys from different floral origins. Significant differences were identified among the studied honey samples from multivariate chemometric methods. The proline content varied among the four types of honeys, with the values decreasing in the order: buckwheat > Codonopsis > sunflower > rapeseed. Rapeseed honeys contained a high level of benzoic acid, while rutin, p-coumaric acid, p-hydroxybenzoic acid were present at relatively high levels in buckwheat honeys. Principal component analysis (PCA) revealed that rapeseed honey could be distinguished from the other three unifloral honeys, and benzoic acid, proline and kaempferol could serve as potential floral markers. Using 18 phenolic compounds and proline the honey samples were satisfactorily classified according to floral origin at 94% correct prediction by linear discriminant analysis (LDA). The results indicated that phenolic compounds and proline were useful for the identification of the floral origin of the four type honeys.

Quantification of melittin and apamin in bee venom lyophilized powder from Apis mellifera by liquid chromatography-diode array detector-tandem mass spectrometry.

A high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) method was developed for simultaneous determination of melittin and apamin in crude bee venom lyophilized powder (CBVLP) as the traditional Chinese medicine possessing specific biological activity. Melittin and apamin were extracted with pure water from CBVLP samples followed by HPLC-DAD-MS/MS analysis. The method was validated to demonstrate its selectivity, linearity, limit of quantification (LOQ), intraday precision, interday precision, accuracy, recovery, matrix effect, and stability. The assay was linear over the concentration ranges of 1-100 and 0.2-25 microg/ml with limit of quantifications (LOQs) of 1.0 and 0.3 microg/ml for melittin and apamin, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 2.2% to 11.4% for intraday repeatability and from 3.2% to 13.1% for interday intermediary precision. The concentrations of endogenous melittin and apamin in CBVLP samples ranged from 46% to 53% and from 2.2% to 3.7% of dry weight, respectively. This rapid, simple, precise, and sensitive method allowed the simultaneous determination of melittin and apamin to evaluate authenticity and quality of CBVLP samples.

Determination of melamine in royal jelly and lyophilized powder using ultrasonically assisted extraction by ion-pair HPLC with UV detection.

A rapid method for the analysis of melamine in royal jelly (RJ) and RJ lyophilized powder (RJLP) was developed using ion-pair RP-HPLC coupled with UV detector. The method utilized an optimized buffer system to avoid the elution of melamine near the column void volume and improve retention of melamine in a generic C8 chromatographic column. In addition, sample preparation included deproteination, ultrasonic-assisted extraction, and cleanup on a mixed-mode cation exchange extraction cartridge. The extraction procedure was optimized with regard to the amount of extraction solvent and the duration of sonication for RJ and RJLP samples. The following criteria were used to validate the HPLC-UV detection method: selectivity, linearity, precision, LOD, and LOQ. Correlation coefficient was higher than 0.999 by applying the linear regression model based on the least square method with a weighting factor (1/x). Precision was evaluated as repeatability and intermediary precision with RSD of less than 15%. The mean percentage recoveries of melamine were varied from 72.5 to 90.5% for RJ and RJLP. This approach will be of particular utility for the evaluation of melamine residue level and routine monitoring of melamine in RJ and RJLP samples.

Gelsedine-type alkaloids: Discovery of natural neurotoxins presented in toxic honey.

The fascinating collection and evaluation of natural products with enormous structural and chemical diversity can contribute to ensure human health and inspire potential drug discovery. We reported the identification of 14-(R)-hydroxy-gelsenicine (HGE), a new component from poisonous honey, which has recently caused multiple serious intoxications and deaths up on consumption. The prevalence, toxicity, toxicokinetics and metabolic profile of HGE were evaluated through in vitro and in vivo analyses. HGE is a very toxic substance and shows significant gender difference with LD50 of 0.125 mg kg-1 and 0.295 mg kg-1 for the female and male mice, respectively. Toxicokinetics test indicates that HGE has good bioavailability in rats, and is metabolized extensively, in which hydroxylation, reduction, N-demethyl ether and glucuronication are the major metabolic pathways. Additionally, HGE shows specific neurotoxicity by enhancing the binding of γ-aminobutyric acid (GABA) to its receptors. We found that flumazenil, a selective antagonist of GABA receptor, could effectively increase the survival of the tested animals, which provides a potential therapy for future clinical applications.

Simultaneous determination of seven fluoroquinolones in royal jelly by ultrasonic-assisted extraction and liquid chromatography with fluorescence detection.

A method for the quantitative determination of seven fluoroquinolone antibacterial agents (FQs) used in beekeeping, viz. ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, danofloxacin, enrofloxacin, and difloxacin, in royal jelly samples was developed on the basis of high performance liquid chromatography with fluorescence detection. Sample preparation included deproteination, ultrasonic-assisted extraction with a mixed inorganic solution of monopotassium phosphate (KH(2)PO(4)) and ethylenediaminetetraacetic acid disodium salt (Na(2)EDTA), and clean-up on a solid-phase extraction cartridge. The extraction procedure was optimized with regard to the amount of inorganic solvent and the duration of sonication for royal jelly as a complicated matrix. Overall recoveries for FQs ranged from 85.9 to 99.1% for royal jelly with standard deviations between 2.79 and 6.27%. Limits of quantification were 2-40 ng/g for seven FQs in royal jelly. A total of 57 real royal jelly samples collected from beekeepers and supermarkets were analyzed. The three most abundant honeybee-use FQs, i. e. ofloxacin, ciprofloxacin, and norfloxacin, were determined in some royal jelly samples in concentrations ranging from 11.9 to 55.6 ng/g. Unexpectedly, however, difloxacin was found at concentrations of about 46.8 ng/g in one sample although it is rarely used in beekeeping. The presented method was successfully applied to quantify FQs in real royal jelly samples.

Determination of tryptophan in bee pollen and royal jelly by high-performance liquid chromatography with fluorescence detection.

A method for tryptophan analysis in bee pollen and royal jelly was developed using HPLC with fluorescence detection. To determine the free tryptophan in bee pollen and royal jelly, ultrasonic extraction was performed using water (pH 6.3)-acetonitrile (10:1, v/v) as extraction solvent. While determining the total tryptophan in these bee products, the method involves alkaline hydrolysis of the proteins with 4 mol/L sodium hydroxide at 110 degrees C for 20 h under anaerobic conditions. The operating conditions for the HPLC analysis were: Symmetry C(18) column (4.6 x 250 mm, 5 microm), 0.1% trifluoroacetic acid-methanol (75:25, v/v) as the mobile phase at a flow rate of 1.0 mL/min at 30 degrees C. The fluorescence detector was operated at an excitation wavelength of 280 nm and an emission wavelength of 340 nm. A linear response (r(2 )> 0.9998) was obtained in the range 0.0625-5.0 microg/mL. The method was successfully applied to the determination of the free and total tryptophan contents in bee pollens, which were 0.069 +/- 0.003 and 2.693 +/- 0.476 mg/g, respectively, while only the total tryptophan was detected in royal jelly, with a content of 1.743 +/- 0.066 mg/g.

Joint Toxicity of Acetamiprid and Co-Applied Pesticide Adjuvants on Honeybees under Semifield and Laboratory Conditions.

The evaluation of adverse effects of pesticides, pesticide adjuvants, and their combination on honeybees is hampered by a lack of colony-level bioassays reflecting productivity and survival over longer term exposure. In the present study, the joint toxicity of acetamiprid and co-applied pesticide adjuvants (N-methyl pyrrolidone [NMP], Silwet L-77, and Triton X-100) to honeybees was determined both in the laboratory and under semifield conditions. The 3 pesticide adjuvants caused no significant acute toxicity to honeybees by themselves; however, in the laboratory tests, they significantly increased the acute contact toxicity of acetamiprid to honeybees. For the semifield tests, in the T2 group (treatment with 5% acetamiprid soluble concentrate [SL] containing 10% Silwet L-77), the mortality of honeybees was significantly higher (p < 0.05) than that of the blank control on the fourth day after application (DAA + 4), that of the T1 group (5% acetamiprid SL containing 10% NMP) on DAA + 4 and DAA + 7 (seventh day after application), and that of the T3 group (5% acetamiprid SL containing 10% Triton X-100) on DAA + 4. Furthermore, the flight intensity in the T2 group on DAA + 7, the colony intensity on DAA + 28 (28th day after application), and the mean areas covered by pupae on DAA + 15 (15th day after application) were significantly lower (p < 0.05) than those of the blank control. Therefore, pesticide adjuvants may be important factors in increasing the toxicity of neonicotinoids to honeybees. Measures should be taken to manage the environmental risk of pesticide adjuvants during the process of formulation development and registration. Environ Toxicol Chem 2019;38:1940-1946. © 2019 SETAC.

A green triple-locked strategy based on volatile-compound imaging, chemometrics, and markers to discriminate winter honey and sapium honey using headspace gas chromatography-ion mobility spectrometry.

A simple and environmentally approach using untargeted imaging of volatile substances combined with chemometrics and markers response was proposed for discriminating different species of honey with headspace gas-chromatography-ion-mobility (HS-GC-IMS). The 3D HS-GC-IMS imaging and their response differences enabled the clear discrimination between winter honey and sapium honey. Principal component analysis (PCA) and partial least-squares discrimination analysis (PLS-DA) were employed to discriminate different honey. Markers of two kinds of honey were identified and confirmed with a user-built imaging database combined with multivariate analysis. Benzaldehyde dimer and phenylacetaldehyde dimer were found to be reliable markers of winter honey, and phenylethyl acetate dimer was of sapium honey. Adulteration identification of the honey samples with different adulteration ratios were subjected to this triple-locked strategy analysis. The results demonstrate that HS-GC-IMS imaging coupled with chemometrics and marker identification is a useful triple-locked strategy to discriminate honey from different floral origins and adulterated honey.

Optimization and application of parallel solid-phase extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry for the determination of 11 aminoglycoside residues in honey and royal jelly.

A robust and sensitive method of solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established and performed for the simultaneous determination of eleven aminoglycosides (AGs) in royal jelly and honey. After sample extraction by a phosphate buffer containing trichloroacetic acid (TCA) and ethylenediaminetetracetic acid disodium salt (Na2EDTA), the extraction solution was subjected to a parallel solid-phase extraction for clean-up prior to the LC-MS/MS analysis. The same method was applied to analyze two completely different matrices, honey and royal jelly. Good sensitivity, repeatability, and recovery were obtained by using the mobile phase without an ion-pairing reagent such as heptafluorobutyric acid (HFBA) or sodium heptanesulfonate. The calibration curves of the honey and royal jelly samples exhibited a good linear response (R2 > 0.99) at six concentrations in the range of 10-1000 µg/mL. The limit of quantification (LOQ) of the AGs ranged from 10 to 25 µg/kg in the honey and from 12.5 to 25 µg/kg in the royal jelly. The recoveries of the AGs for the honey and royal jelly samples were in the range of 79.48% to 108.95% and 74.61% to 113.70% respectively and the relative standard deviations (RSDs) were between 1.23% and 9.59%, and between 1.51% and 9.98%, respectively. The proposed approach has been allowed in China as a reference method for the simultaneous determination of eleven AGs in honey and royal jelly.

Toxicokinetics of HT-2 Toxin in Rats and Its Metabolic Profile in Livestock and Human Liver Microsomes.

The lack of information on HT-2 toxin leads to inaccurate hazard evaluations. In the present study, toxicokinetic studies of HT-2 toxin were investigated following intravenous (iv) and oral administration to rats at dosages of 1.0 mg per kilogram of body weight. After oral administration, HT-2 toxin was not detected in plasma, whereas its hydroxylated metabolite, 3'-OH HT-2 was identified. Following iv administration, HT-2 toxin; its 3'-hydroxylated product; and its glucuronide derivative, 3-GlcA HT-2, were observed in plasma, and the glucuronide conjugate was the predominant metabolite. To explore the missing HT-2 toxin in plasma, metabolic studies of HT-2 toxin in liver microsomes were conducted. Consequently, eight phase I and three phase II metabolites were identified. Hydroxylation, hydrolysis, and glucuronidation were the main metabolic pathways, among which hydroxylation was the predominant one, mediated by 3A4, a cytochrome P450 enzyme. Additionally, significant interspecies metabolic differences were observed.

Optimized determination method for trans-10-hydroxy-2-decenoic acid content in royal jelly by high-performance liquid chromatography with an internal standard.

An optimized reversed-phase high-performance liquid chromatography method was developed to detect the trans-10-hydroxy-2-decenoic acid (10-HDA) content in royal jelly cream and lyophilized powder. The sample was extracted using absolute ethanol. Chromatographic separation of 10-HDA and methyl 4-hydroxybenzoate as the internal standard was performed on a Nova-pak C18 column. The average recoveries were 95.0-99.2% (n = 5) with relative standard deviation (RSD) values of 1.3-2.1% for royal jelly cream and 98.0-100.0% (n = 5) with RSD values of 1.6-3.0% for lyophilized powder, respectively. The limits of detection and quantitation were 0.5 and 1.5 mg/kg, respectively, for both royal jelly cream and lyophilized powder. The method was validated for the determination of practical royal jelly products. The concentration of 10-HDA ranged from 1.26 to 2.21% for pure royal jelly cream samples and 3.01 to 6.19% for royal jelly lyophilized powder samples. For 30 royal jelly products, the 10-HDA content varied from not detectable to 0.98%.

Simultaneous determination of nitroimidazole residues in honey samples by high-performance liquid chromatography with ultraviolet detection.

A rapid and sensitive high-performance liquid chromatography (LC) method was developed for the simultaneous determination of metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), tinidazole (TNZ), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI) in honey. After extraction with ethyl acetate and evaporation, the residue containing the nitroimidazoles was dissolved in ethyl acetate-hexane and subjected to solid-phase extraction cleanup by amine extraction columns. The effluent was evaporated to dryness, and residues were dissolved and determined by LC with an ultraviolet detector set at 315 nm. The limits of detection were 1.0-2.0 ng/g for MNZ, DMZ, RNZ, TNZ, and HMMNI in honey. Average recoveries ranged from 71.5-101.4% in honey fortified at 10, 20, 50, and 100 ng/g. The method was validated for the analysis of real honey samples.

The Microbial Community Dynamics during the Vitex Honey Ripening Process in the Honeycomb.

The bacterial and fungal communities of vitex honey were surveyed by sequencing the 16S rRNA gene and the internal transcribed spacer region of ribosomal DNA. Vitex honey samples were analyzed at different stage of ripening; the vitex flower was also analyzed, and the effect of the chemical composition in the experimental setup was assessed. The results confirmed the presence of dominant Bacillus spp. as the dominant bacterial in honey, and yeast related genera was the main fungal in the honey, respectively. Lactococcus and Enterococcus were detected for the first time in honey. The proportion of most of the fungal community decreased during the honey ripening process. Multivariate analyses also showed that the fungal community of 5, 10, and 15 days honey samples tended to cluster together and were completely separated from the 1 day honey sample. The change in the fungal community showed a correlation with the variation in the chemical components, such as moisture and phenolic compounds. Together, these results suggest that ripening of honey could change its microbial composition, and decrease the potential risk of microbiology.

Development and validation of a multiclass method for the quantification of veterinary drug residues in honey and royal jelly by liquid chromatography-tandem mass spectrometry.

The aim of this study was to develop an analytical method for the analysis of a wide range of veterinary drugs in honey and royal jelly. A modified sample preparation procedure based on the quick, easy, cheap, effective, rugged and safe (QuEChERS) method was developed, followed by liquid chromatography tandem mass spectrometry determination. Use of the single sample preparation method for analysis of 42 veterinary drugs becomes more valuable because honey and royal jelly belong to completely different complex matrices. Another main advantage of the proposed method is its ability to identify and quantify 42 veterinary drugs with higher sensitivity than reference methods of China. This work has shown that the reported method was demonstrated to be convenient and reliable for the quick monitoring of veterinary drugs in honey and royal jelly samples.

Comprehensive Analysis of Tiamulin Metabolites in Various Species of Farm Animals Using Ultra-High-Performance Liquid Chromatography Coupled to Quadrupole/Time-of-Flight.

Tiamulin is an antimicrobial widely used in veterinary practice to treat dysentery and pneumonia in pigs and poultry. However, knowledge about the metabolism of tiamulin is very limited in farm animals. To better understand the biotransformation of tiamulin, in the present study, in vitro and in vivo metabolites of tiamulin in rats, chickens, swine, goats, and cows were identified and elucidated using ultra-high performance liquid chromatography coupled to quadrupole/time-of-flight. As a result, a total of 26 metabolites of tiamulin, identified in vitro and in vivo, and majority of metabolites were revealed for the first time. In all farm animals, tiamulin undergoes phase I metabolic routes of hydroxylation in the mutilin part (the ring system), S-oxidation and N-deethylation on side chain, and no phase II metabolite was detected. Among these, 2ß- and 8α-hydroxylation and N-deethylation were the main metabolic pathways of tiamulin in farm animals. In addition, we have put forward that 8a-hydroxy-tiamulin and 8a-hydroxy-N-deethyl-tiamulin could be hydroxylated into 8a-hydroxy-mutilin, the marker residue of tiamulin in swine. Furthermore, a significant interspecies difference was observed on the metabolism of tiamulin among various farm animals. The possible marker residues for tiamulin in swine were 8α-hydroxy-tiamulin, N-deethyl-tiamulin, and 8α-hydroxy-N-deethyl-tiamulin, which were consistent with the hypothesis proposed by the European Agency for the Evaluation of Medicinal Products. However, results in present study indicated that three metabolites (2ß-hydroxy-tiamulin, N-deethyl-tiamulin, and 2ß-hydroxy-N-deethyl-tiamulin) of tiamulin in chickens had larger yields, which implied that 2ß-hydroxy-mutilin or N-deethyl-tiamulin was more likely to be regarded as the potential marker residue of tiamulin in chickens.

Strategy for comparative untargeted metabolomics reveals honey markers of different floral and geographic origins using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry.

Honey discrimination based on floral and geographic origins is limited by the ability to determine reliable markers because developing hypothetical substances in advance considerably limits the throughput of metabolomics studies. Here, we present a novel approach to screen and elucidate honey markers based on comparative untargeted metabolomics using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap). To reduce metabolite information losses during sample preparation, the honey samples were dissolved in water and centrifuged to remove insoluble particles prior to UHPLC-Q-Orbitrap analysis in positive and negative electrospray ionization modes. The data were pretreated using background subtraction, chromatographic peak extraction, normalization, transformation and scaling to remove interferences from unwanted biases and variance in the experimental data. The pretreated data were further processed using principal component analysis (PCA) and a three-stage approach (t-test, volcano plot and variable importance in projection (VIP) plot) to ensure marker authenticity. A correlation between the molecular and fragment ions with a mass accuracy of less than 1.0ppm was used to annotate and elucidate the marker structures, and the marker responses in real samples were used to confirm the effectiveness of the honey discrimination. Moreover, we evaluated the data quality using blank and quality control (QC) samples based on PCA clustering, retention times, normalized levels and peak areas. This strategy will help guide standardized, comparative untargeted metabolomics studies of honey and other agro-products from different floral and geographic origins.