Guojun Dai

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection.

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.

Quantitative analysis of amoxicillin, its major metabolites and ampicillin in eggs by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this present study, we developed a simple, rapid and specific method for the quantitative analysis of the contents of amoxicillin (AMO), AMO metabolites and ampicillin (AMP) in eggs. This method uses a simple liquid-liquid extraction with acetonitrile followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimized method has been validated according to requirements defined by the European Union and Food and Drug Administration. Extraction recoveries of the target compounds from the egg at 5, 10 and 25 μg/kg were all higher than 80%, with relative standard deviations not exceeding 10.00%. The limits of quantification in eggs were below the maximum residue limits (MRLs). The decision limits (CCα) ranged between 11.1 and 11.5 μg/kg, while detection capabilities (CCβ) from 12.1 to 13.0 μg/kg. These values were very close to the corresponding MRLs. Finally, the new approach was successfully verified for the quantitative determination of these analytes in 40 commercial eggs from local supermarkets.

Simultaneous determination of amoxicillin and ampicillin in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection using pre-column derivatization.

A sensitive and robust method is presented for the simultaneous determination of amoxicillin (AMO) and ampicillin (AMP) in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD). This method used a simple liquid-liquid extraction of the samples with acetonitrile and dichloromethane as precipitation of proteins and extraction solvent. AMO and AMP reacted with salicylaldehyde to form fluorescent derivatives, which were then analyzed with RP-HPLC-FLD. Separation was carried out on an Athena C18 column with a mobile phase consisting of 0.01 M potassium dihydrogen phosphate, adjusted to pH 5.5 by 2M potassium hydroxide and acetonitrile. The detector response was linear over the tested concentration range from 5.0 to 800 ng/mL for AMO and AMP. The recovery values ranged from 78.4 to 88.7% for AMO and from 77.6 to 82.0% for AMP. The limits of detection were 1.2 for AMO and 0.4 µg/kg for AMP. The limits of quantification were 3.9 for AMO and 1.5 µg/kg for AMP. The corresponding intra-day and inter-day variation (relative standard deviation) were found to be less than 9.6 and 14.8%, respectively.

Determination and depletion of amoxicillin residues in eggs

Eggs were used to study the determination and depletion of amoxicillin (AMO) residues after oral dosing hens (25.0 mg kg–1, 50.0 mg kg–1 body weight), once daily for five days. A reverse-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed to determine AMO residues in albumen, yolk and whole egg. By using pre-column derivatisation, an improved liquid–liquid extraction procedure was developed for sample preparation. AMO were extracted from eggs with acetonitrile. The extract solution was extracted using saturated methylene chloride. The supernatant was reacted with salicylaldehyde under acidic and heating conditions. Limits of detection (LODs) were 1.2 ng g–1 and limits of quantification (LOQs) were 3.9 ng g–1 for AMO. Recoveries of AMO from samples fortified at levels of 5.0–125.0 ng g–1 ranged from 79.1% to 88.5% in albumen, 78.6–83.6% in yolk and 78.3–85.1% in whole egg, with coefficients of variation of ≤7.3%. The maximum concentrations of AMO in albumen, yolk and whole egg were found to occur at 1.5, 2.5, 1.5 days after withdrawal of medication respectively. AMO was not detectable in albumen at 7.5 days after final administration of AMO, at 10.5–11.5 days in yolk and 10.5 days in whole egg after administration of two oral doses. The method was applied during the residue study of AMO in order to formulate a reasonable withdrawal period to ensure food safety.

Polymorphisms of the Myostatin Gene and Its Relationship with Reproduction Traits in the Bian Chicken

Myostatin, or growth and differentiation factor 8, is a member of the transforming growth factor-β superfamily; it functions as a negative regulator of skeletal muscle development and growth in mammals. In this study, single nucleotide polymorphisms in the 5′ regulatory region and exon 1 of the myostatin gene were detected by PCR–SSCP in the Bian, Jinghai, Youxi, and Arbor Acre chickens, and the associations of the polymorphisms with reproduction traits were analyzed. Seven SNPs (A326G, C334G, C1346T, G1375A, A1473G, G1491A, and G2283A) were found in the myostatin gene. Association analysis showed that the G2283A were significantly associated with reproduction traits. Bian chickens of the GG genotype had a greater age at first egg than those of the GA and AA genotypes (P < 0.01). Correspondingly, Bian chickens of the GA and AA genotypes had larger egg number at 300 days than those of the GG genotype (P < 0.05 and P < 0.01, respectively). Bian chickens of the AA genotype had significantly higher body weight at 300 days than those of the GG genotype (P < 0.05). These results suggested that the myostatin gene may have certain effects on reproduction traits other than merely as a negative regulator of skeletal muscle development and growth in mammals previously reported.

Residue depletion of ampicillin in eggs.

A residue depletion study of ampicillin (AMP) was performed after oral dosing (60.0 mg/kg and 120.0 mg/kg body weight once a day for 5 days) to laying hens, through the use of reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) to achieve detection of ampicillin residue in eggs. Limit of detection was 0.5 ng/g, and limit of quantitation was 1.2 ng/g for ampicillin. Extraction recoveries of ampicillin from samples fortified at 5.0-125.0 ng/g levels ranged from 77.5% to 84.6% in albumen, 77.9% to 87.5% in yolk, and 77.9% to 88.6% in whole egg, with coefficients of variation ≤ 9.3%. The maximum concentrations of ampicillin in albumen, yolk, and whole egg were detected at 1, 2, and 1 day after the last administration of ampicillin, respectively. Ampicillin was not detectable in albumen at day 9 of withdrawal time, at day 10 and 11 in yolk, and day 9 and 11 in whole egg at each of those 2 dose levels. The theoretical withdrawal time of AMP in whole egg was 6.730 and 7.296 days for 60 and 120 mg/kg oral dosage, respectively. This method also proved to be suitable as a rapid and reliable method for the determination of ampicillin in other poultry eggs.

Development and comparison of HPLC-MS/MS and UPLC-MS/MS methods for determining eight coccidiostats in beef

A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining eight coccidiostat (halofuginone, lasalocid, maduramicin, monensin, narasin, nigericin, robenidine and salinomycin) residues in beef were developed and compared. Samples were extracted with a mixture of acetic acid, acetonitrile and ethyl acetate and were then purified on a C18 solid-phase extraction (SPE) column. The purified samples were analyzed by HPLC-MS/MS and UPLC-MS/MS, using 0.1% formic acid-water solution (A) and pure methanol (B) as the mobile phase. The samples were fractionated on a C18 column using different gradient elution procedures, followed by qualitative analysis using a mass spectrometer operated in multiple reaction monitoring (MRM) mode with positive electrospray ionization; the external standard method was used for quantitation. At spiked levels that ranged from the limit of quantification (LOQ) to 100 μg/kg, the average recoveries were 71.96%-100.32% and 71.24%-89.24%, with relative standard deviations (RSDs) of 2.65%-12.38% and 2.98%-14.86% for UPLC-MS/MS and HPLC-MS/MS, respectively. The limits of detection (LODs) and LOQs of the eight coccidiostats were 0.14-0.32 μg/kg and 0.43-1.21 μg/kg, respectively, for UPLC-MS/MS analysis and 0.16-0.58 μg/kg and 0.53-1.92 μg/kg, respectively, for HPLC-MS/MS analysis. Both methods had good accuracy and precision, but UPLC-MS/MS had higher sensitivity than HPLC-MS/MS.

Bioinformatics Analysis of SNPs in IL-6 Gene Promoter of Jinghai Yellow Chickens

The proinflammatory cytokine, interleukin-6 (IL-6), plays a critical role in many chronic inflammatory diseases, particularly inflammatory bowel disease. To investigate the regulation of IL-6 gene expression at the molecular level, genomic DNA sequencing of Jinghai yellow chickens (Gallus gallus) was performed to detect single-nucleotide polymorphisms (SNPs) in the region −2200 base pairs (bp) upstream to 500 bp downstream of IL-6. Transcription factor binding sites and CpG islands in the IL-6 promoter region were predicted using bioinformatics software. Twenty-eight SNP sites were identified in IL-6. Four of these 28 SNPs, three [−357 (G > A), −447 (C > G), and −663 (A > G)] in the 5′ regulatory region and one in the 3′ non-coding region [3177 (C > T)] are not labelled in GenBank. Bioinformatics analysis revealed 11 SNPs within the promoter region that altered putative transcription factor binding sites. Furthermore, the C-939G mutation in the promoter region may change the number of CpG islands, and SNPs in the 5′ regulatory region may influence IL-6 gene expression by altering transcription factor binding or CpG methylation status. Genetic diversity analysis revealed that the newly discovered A-663G site significantly deviated from Hardy-Weinberg equilibrium. These results provide a basis for further exploration of the promoter function of the IL-6 gene and the relationships of these SNPs to intestinal inflammation resistance in chickens.

Quantitative analysis of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in eggs via liquid chromatography-electrospray ionization tandem mass spectrometry

A widely applicable method involving liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (LC-ESI/MS/MS) was developed for the simultaneous determination of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v) and defatted with hexane saturated with acetonitrile. The analysis was carried out on a mass spectrometer via an electrospray interface operated in the positive and negative ionization modes using deuterated chloramphenicol-d5 as the internal standard. The limits of detection and limits of quantification were 0.04-0.5 μg/kg and 0.1-1.5 μg/kg in eggs, respectively. The average recoveries of the four analytes from egg samples were 90.84-108.23%, with relative standard deviations of less than 9.61%. The corresponding intra-day and inter-day variations were found to be less than 8.11% and 11.30%, respectively. Finally, the new approach was successfully applied to the quantitative determination of these analytes in 50 commercial eggs from local supermarkets.

Quantification of piperazine in chicken and pig tissues by gas chromatography–electron ionization tandem mass spectrometry employing pre-column derivatization with acetic anhydride

This paper describes a novel method that combines acetic anhydride derivatization with gas chromatography-electron ionization tandem mass spectrometry (GC-EI/MS/MS) for the sensitive and selective determination of piperazine in chicken and pig tissues. Samples were extracted using an accelerated solvent extraction (ASE) apparatus, purified by solid-phase extraction (SPE) and derivatized with acetic anhydride. This optimized method was validated according to the requirements defined by the European Union and the Food and Drug Administration. At the limit of quantification (LOQ) spiked levels of 50.0, 100.0, 500.0, 1000.0 and 2000.0μg/kg, the average recoveries of piperazine in chicken and pig tissues were 77.46-96.26%, with relative standard deviations (RSDs) of 1.55-6.64%. The intra-day RSDs were 1.39-5.92%, and the inter-day RSDs were 2.24-8.39%. The limits of detection (LODs) and the LOQs were 1.4-1.6μg/kg and 4.8-5.2μg/kg, respectively. The decision limits (CCα) were 102.02-105.17μg/kg, and the detection capabilities (CCβ) were 104.03-109.09μg/kg. Finally, the new approach was verified for the quantitative determination of piperazine in 30 commercial chicken and pig tissues from local supermarkets.