Liming Wu

Floral classification of honey using liquid chromatography-diode array detection-tandem mass spectrometry and chemometric analysis.

A high performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) method for the floral origin traceability of chaste honey and rape honey samples was firstly presented in this study. Kaempferol, morin and ferulic acid were used as floral markers to distinguish chaste honey from rape honey. Chromatographic fingerprinting at 270 nm and 360 nm could be used to characterise chaste honey and rape honey according to the analytical profiles. Principal component analysis (PCA), partial least squares (PLS), partial least squares-discrimination analysis (PLS-DA) and soft independent modeling of class analogy (SIMCA) were applied to classify the honey samples according to their floral origins. The results showed that chaste honey and rape honey could be successfully classified by their floral sources with the analytical methods developed through this study and could be considered encouraging and promising for the honey traceability from unifloral or multifloral nectariferous sources.

Geographical traceability of propolis by high-performance liquid-chromatography fingerprints

A rapid fingerprint method was developed for investigating and inferring geographical origin of Chinese propolis by using high performance liquid chromatography-ultraviolet detection (HPLC-UV). 120 samples were analyzed from 17 different locations of 10 provinces of China in this study. In the HPLC chromatograms, eight major compounds were identified as flavonoids, including rutin, myricetin, quercetin, kaempferol, apigenin, pinocembrine, chrysin and galangin. Both correlation coefficient of similarity in chromatograms and relative peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. Our results revealed that the presence or absence of specific peaks and similarity evaluation in simulative mean chromatograms among different regions could efficiently identify and distinguish Chinese propolis from different geographical origins.

Quantitative determination of juvenile hormone III and 20-hydroxyecdysone in queen larvae and drone pupae of Apis mellifera by ultrasonic-assisted extraction and liquid chromatography with electrospray ionization tandem mass spectrometry

In this paper, a method for the rapid and sensitive analysis of juvenile hormone III (JH III) and 20-hydroxyecdysone (20E) in queen larvae and drone pupae samples was presented. Ultrasound-assisted extraction provided a significant shortening of the leaching time for the extraction of JH III and 20E and satisfactory sensitivity as compared to the conventional shake extraction procedure. After extraction, determination was carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in electrospray ionization positive ion mode via multiple reaction monitoring (MRM) without any clean-up step prior to analysis. A linear gradient consisting of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid, and a ZORBAX SB-Aq column (100 mm × 2.1 mm, 3.5 μm) were employed to obtain the best resolution of the target analytes. The method was validated for linearity, limit of quantification, recovery, matrix effects, precision and stability. Drone pupae samples were found to contain 20E at concentrations of 18.0 ± 0.1 ng/g (mean ± SD) and JH III was detected at concentrations of 0.20 ± 0.06 ng/g (mean ± SD) in queen larvae samples. This validated method provided some practical information for the actual content of JH III and 20E in queen larvae and drone pupae samples.

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.

Identification of monofloral honeys using HPLC–ECD and chemometrics

A total of 77 jujube, longan and chaste honey samples were collected from 18 different areas of China. Thirteen types of phenolic acids in the honey samples were analysed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Moreover, HPLC-ECD fingerprints of the monofloral honey samples were established. From the analysis of the HPLC-ECD fingerprints, common chromatography peak information was obtained, and principal component analysis and discriminant analysis were performed using selected common chromatography peak areas as variables. By comparing with phenolic acids as variables, using a chemometric analysis which is based on the use of common chromatography peaks as variables, 36 honey samples and 41 test samples could be correctly identified according to their floral origin.

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.

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.

2-Acetylfuran-3-Glucopyranoside as a Novel Marker for the Detection of Honey Adulterated with Rice Syrup

The determination of honey authenticity is of importance to ensure its quality and safety. There is an urgent need of effective methods to detect adulterated honey. A simple, rapid, and effective HPLC-DAD method was developed to detect honey adulteration by rice syrup, using a characteristic compound from rice syrup, which is presently difficult to detect by current analytical methods. The characteristic compound was identified as 2-acetylfuran-3-glucopyranoside (AFGP) by MS and NMR. Based on HPLC analyses, the average concentration of AFGP was 92 ± 60 mg/kg in rice syrup. However, AFGP was not detected in any of the natural honey samples, so it could be used as a marker for the detection of honey adulteration by rice syrup. The developed method enabled a rapid detection of honey samples adulterated with 10% rice syrup. Using the developed method, 16 out of 186 honey samples from some markets were found to be adulterated with rice syrup.

Hydrophilic interaction chromatography/tandem mass spectrometry for the determination of melamine in royal jelly and royal jelly lyophilized powder

Melamine has become the focus of attention for the possible occurrence of nephrolithiasis and associated deaths, because it was added to foods to increase the apparent protein content by unethical manufacturers. An analytical method based on hydrophilic interaction chromatography/tandem mass spectrometry (HILIC-MS/MS) was developed and validated for the determination of melamine in the royal jelly (RJ) and royal jelly lyophilized powder (RJLP). Trace of melamine was extracted from the RJ and RJLP by ultrasonic-assisted extraction followed by clean-up procedure using mixed-mode cation exchange (MCX) solid phase extraction and separated on a hydrophilic interaction chromatography (HILIC) analytical column with acetonitrile/5mM ammonium acetate buffer (88:12, v/v) as mobile phase. Detection was carried out by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The chromatographic separation was obtained within 5min and was linear in the concentration range of 0.01-8microg/mL in RJ and 0.05-10microg/mL in RJLP for melamine. The mean extraction recoveries for melamine were ranged from 89.6 to 100.4%. Method validation parameters were evaluated such as linearity, selectivity, precision, carryover and recovery, giving results within the acceptable range. The proposed method was successfully applied to the quantitation of melamine in RJ and RJLP. This approach will be of particular utility for the evaluation of melamine residue level and routine monitor of melamine in RJ and RJLP samples.

Analysis of coenzyme Q10 in bee pollen using online cleanup by accelerated solvent extraction and high performance liquid chromatography

A method for the determination of coenzyme Q10 in bee pollen has been developed applying an online cleanup of accelerated solvent extraction and using environmentally acceptable organic solvents. The extracted samples were analysed by high performance liquid chromatography with diode array detection. The optimised method employed 10 mL extraction cells, 1g sample size, absolute ethanol as extraction solvent, 80°C of extraction temperature, one extraction cycle, 5 min of static time, Cleanert Alumina-N as sorbent and 60% flush volume. The method was validated by means of an evaluation of the matrix effects, linearity, limit of detection (LOD) and quantification (LOQ), trueness, precision and stability. The assay was linear over the concentration range of 0.25-200mg/L and the LOD and LOQ were 0.16 and 0.35 mg/kg, respectively. The recoveries were above 90%. The inter- and intra-day precision was below 6.3%. The method has been successfully applied to the analysis of bee pollen samples. For 20 bee pollen products, the coenzyme Q10 content varied from not detectable to 192.8 mg/kg.